A highly dynamic mononuclear non-heme iron enzyme for the two-step isonitrile biosynthesis

Domain: neuroscience
Published: 2026-01-26
Editorial outcome: accept

Abstract
The recent discovery of the isonitrile biosynthetic enzyme ScoE expanded the catalytic repertoire of the Fe(II)/αKG-dependent dioxygenase enzyme family. ScoE synthesizes an isonitrile functional group from a glycyl-fatty acid adduct, with both the isonitrile nitrogen and carbon atoms coming from the glycyl moiety. This challenging chemistry cannot be performed in a single step. Instead, the mechanism appears to require two half reactions, each involving αKG cleavage to generate a highly reactive iron-oxygen species. Here, we report sixteen crystal structures that provide snapshots along the reaction trajectory of Rv0097, a ScoE homolog from Mycobacterium tuberculosis. These structures, which are both of wild-type and Rv0097 variants, include a substrate 3-((carboxymethyl)amino)decanoic acid (CADA)-bound structure, an αKG-bound structure, and a structure with both CADA and αKG bound. These structural data reveal how Rv0097 employs conformational rearrangements to protect the unstable CADA-reaction intermediate that is formed in the first half reaction while swapping out αKG cleavage products for a second molecule of αKG. Additionally, these structures, together with data from site-directed mutagenesis, provide insight into Rv0097’s preference for substrates with long alkyl chains, potentially facilitating efforts to re-engineer ScoE/Rv0097 to synthesize isonitrile functional groups on a wider range of small molecules.

Introduction
The isonitrile functional group, with its zwitterionic character, is the subject of diverse chemical reactivities in organic syntheses. It acts as a nucleophile, an electrophile, a carbene, a radical acceptor, and a metal-binding ligand1–4. In nature, isonitrile can be found as a bioactive warhead. Since the initial discovery and identification of xanthocillin in 19505, hundreds of isonitrile-containing natural products have been isolated that participate in many biological processes, such as virulence, metal acquisition, and detoxification6–8. To date, however, only two isonitrile biosynthetic routes are known. The first route involves enzymes from the isonitrile synthase (IsnA) family that synthesize isonitrile by condensation of the α-nitrogen atom from L-Tyr/L-Trp and a carbon atom from ribulose-5-phosphate (Supplementary Fig. 1A)9. The second route was reported recently in a study of conserved actinobacteria gene clusters involved in the biosynthesis of isonitrile lipopeptides (INLP)10. In the INLP biosynthetic gene cluster ScoA-E found in Streptomyces coeruleorubidus, ScoE is a non-heme Fe(II)/α-ketoglutarate (αKG)-dependent enzyme that catalyzes isonitrile formation by converting the glycyl moiety of a single substrate, (R)−3-((carboxymethyl)amino)butanoic acid (CABA), to an isonitrile group in the product, (R)−3-isocyanobutanoic acid (INBA) (Fig. 1A)11. In a homologous gene cluster found in Mycobacterium tuberculosis H37Rv (Mtb), Rv0097 performs isonitrile biosynthesis on the glycyl moiety of substrates with long alkyl chains12, including one with a 10-carbon fatty-acyl chain (C10), 3-((carboxymethyl)amino)decanoic acid (CADA), that is transformed to 3-isocyanodecanoic acid (INDA) (Fig. 1B)12. These ScoE/Rv0097-catalyzed isonitrile syntheses expanded the chemical repertoire of enzymes in the Fe(II)/αKG-dioxygenase superfamily.

Recently, we and others have characterized the ScoE-catalyzed reaction using both biochemical and structural approaches13–16. As a member of the non-heme Fe(II)-αKG-dependent dioxygenase enzyme family, ScoE relies on the cleavage of an Fe(II)-bound molecule of αKG by molecular oxygen to form a highly reactive iron-oxygen species, characterized in several family members to be an Fe(IV)-oxo intermediate17–20. This highly reactive Fe(IV)-oxo species can initiate a radical-based reaction by hydrogen atom abstraction from the substrate. For ScoE, we established the reaction stoichiometry and showed that two equivalents of αKG and molecular oxygen are required for every equivalent of CABA consumed and INBA produced13. Cleavage products of αKG: succinate and CO2 are also produced13. We later reported putative intermediates and shunt products detected from the ScoE reaction and proposed a two half-reaction mechanism for isonitrile formation (Supplementary Fig. 1B)14. In this mechanism, the first half-reaction desaturates CABA using one equivalent of αKG and O2 to form an imine intermediate (imine-CABA), which is then decarboxylated to form the isonitrile product by the second half-reaction using the second equivalents of αKG and O214. Although synthesizing an isonitrile group from a glycyl moiety is challenging, nature appears to overcome this hurdle through two sequential oxidation reactions mediated by two Fe(IV)-oxo intermediates. ScoE and its homologs are not alone in catalyzing multiple oxidative events in one catalytic cycle. Multifunctional Fe(II)/αKG-dependent dioxygenases are reported in the biosynthesis of fungal natural products that involve up to four rounds of oxidation reactions (Supplementary Fig. 2)21–24. We are therefore interested in uncovering nature’s design principles that allow for multiple reactions on a single substrate to afford chemically challenging conversions.

To accomplish two half-reactions in one catalytic cycle, ScoE faces several challenges in its enzyme design. First, αKG and CABA are highly structurally similar, thus the enzyme active site would need to differentiate between these two co-substrates. Second, the αKG and CABA binding sites must be close to each other for catalysis but have separate routes for entry and exit. Separate routes allow the enzyme to hold onto the hydrolysis-prone imine-CABA intermediate while exchanging succinate and CO2 for a second molecule of αKG. Our previous ScoE structural studies13 led to the proposal of two conformational changes for the binding of co-substrates (Supplementary Fig. 3); a small conformational change of K193 swinging into the active site to bind CABA, and a large (~10 Å) conformational change of R157/H299 loops flipping toward and away from the active site to allow the binding of αKG molecules by R157/H299 and release of reaction products. Because no structure of ScoE has been obtained that shows αKG bound in a catalytically-relevant fashion, the latter proposal is based on a tartrate-bound ScoE structure in which R157 was observed in the active site interacting with tartate15. Collectively, structural data from our lab13 and others15 started to unravel how ScoE performs isonitrile synthesis by the two half-reaction mechanism, but we are missing key pieces of the puzzle, including a structure with αKG bound in a catalytically competent position.

In this work, we shift our focus to Rv0097, an enzyme homologous to ScoE (45% identity, 61% similarity) from the gene cluster Rv0097-Rv0101 found in Mtb. This gene cluster biosynthesizes an INLP involved in metal transport in Mtb, although the identity of the metal is still in debate8,25,26. Genetic studies showed that the Rv0097-Rv0101 gene cluster is required for Mtb survival in mouse macrophages27, and perturbations of the rv0097 gene led to attenuated Mtb growth and survival in mice28. Therefore, a detailed mechanistic understanding of Rv0097 could contribute to structure-guided and mechanism-based antibiotics design for tuberculosis treatments. Our biochemical studies suggested that Rv0097 recognizes substrates with a long alkyl chain (C8-C16) with a preference of >C10, whereas ScoE recognizes short-chain substrates (C4-C8) with a preference of C412. Since the substrates of ScoE and Rv0097 only differ in alkyl chain lengths, we propose the same two half-reaction mechanism applies for Rv0097 (Fig. 1C, Supplementary Fig. 3).

Here, we add to the structural data on ScoE11,13,15,16 and Rv009726 by obtaining a series of atomic-resolution crystal structures of Rv0097 in different states that enabled us to uncover the enzyme design principles that allow for the control of substrate specificity and allow for two oxidative events in one catalytic cycle. Based on our structural data, we propose a multi-layered conformational gating mechanism for the binding and exit of co-substrates and co-products that involve multiple sites of the enzyme. Our proposed mechanism is further supported by structure-guided mutagenesis and structural and biochemical studies on the variants.

Results
We started by determining the 1.28-Å resolution crystal structure of Rv0097 apoprotein (apo) (structure 1 in Supplementary Table 1-2) by molecular replacement (MR) using our ScoE structure (PDB: 6XOJ)13 (Fig. 2A). Rv0097 and ScoE share high structural similarity (all atom RMSD = 2.159 Å, Cα RMSD = 1.987 Å), both sharing the same double-stranded β-helix fold, also known as the jelly roll or cupin fold, which is common among enzymes in the family of Fe(II)/αKG-dependent dioxygenases29. The asymmetric unit (ASU) contains a homodimer that is held together by extensive hydrophobic interactions and a helix-loop region (Supplementary Fig. 4). Next to the dimer interface region are the substrate-binding site and Fe site (Fig. 2A). A conformationally flexible region (Fig. 2A) contains conserved His and Arg residues (R157/H299 in ScoE; R122/H264 in Rv0097). In the Fe site, we observe electron density for a metal that is ligated to the His2Asp1 triad, two water molecules, and an acetate ion (Fig. 2B). Since an ethylenediaminetetraacetic acid (EDTA) chelation step was included in protein purification to prevent adventitious binding of metals (see Methods) and X-ray fluorescence of the resulting protein crystal indicated successful transition metal chelation, we modeled a Mg2+ ion from the crystallization solution into this density (Fig. 2B). Alternative modeling of a water molecule into this density results in positive difference density (Supplementary Fig. 5) and modeling of heavy metals (such as Fe or Zn) results in negative difference density (Supplementary Fig. 5). In the absence of CADA, an acetate ion is bound in the substrate-binding site. This acetate hydrogen bonds to Y70 and K158 side chains, and to a chain of water molecules that lead to the Mg2+ ion (Fig. 2B).

A pair of residues in the substrate-binding pocket gates substrate binding and specificity
Leading to the active site in the putative substrate-binding pocket that is occupied by acetate in structure 1, we observe two rotamer conformations of the F102 residue (F102 and F102’) (Fig. 2C). Structure refinements with only one of the two F102 conformations led to significant negative difference density at the modeled rotamer and positive difference density at the unmodeled rotamer, suggesting the presence of both conformations at partial occupancies (Fig. 2C). An omit map also shows the density for both F102 conformations (Fig. 2C). Refined occupancy suggests both rotamers are present roughly half of the time (0.48/0.52). Adjacent to this F102 residue is G204, a residue that was previously identified to be non-conserved across homologs (Gly in Rv0097 and MmaE, but Phe in ScoE (F239))12. Structural overlay of Rv0097 with ScoE (PDB: 6XOJ) shows steric clashes between the F102’ rotamer of Rv0097 and F239 of ScoE, indicating that the alternate conformation of F102 is enabled by the lack of a side chain for G204 as opposed to its counterpart residue F239 in ScoE (Fig. 2D).

To examine the role of this F102/G204 residue pair in substrate binding in Rv0097, we obtained a 1.12-Å resolution crystal structure with Fe(II) and CADA co-crystallized (Fig. 2E, structure 2 in Supplementary Table 1-2). We note here that we used racemic-CADA for co-crystallization due to substantial costs and time investments it requires to synthesize the enantiopure (R)-CADA26. We find only the (R) isomer bound in the structure, indicating the preference of the enzyme for (R)-CADA over (S)-CADA, as has been previously reported26. This Fe(II)-CADA bound structure is highly similar to the previously reported Rv0097 structure with Fe(II) and CADA bound26 (PDB: 8KHT) (all atom RMSD = 1.126 Å, Cα RMSD = 0.613 Å). Since CADA has a polar moiety that is identical to the polar moiety of ScoE’s substrate CABA and a nonpolar alkyl chain that is longer (seven carbon units) than the single carbon unit (methyl) moiety in CABA, Rv0097 has a binding site that is both polar and nonpolar (Fig. 2E). The conformation and interactions of the polar part of CADA in Rv0097 are very similar to the conformation and interactions of CABA observed in ScoE (Supplementary Fig. 6). The two negatively charged carboxylates of CADA form charge-charge interactions with side chains of K158 and R275 (Fig. 2E). One carboxylate additionally forms a hydrogen bond with Y70. The Y65 side chain hydrogen bonds to the nitrogen of the glycine moiety of CADA. The long alkyl chain of CADA sits snugly in the substrate-binding site, stabilized by hydrophobic interactions with F95, M101, F102, P153, H156, C201, T203, and G204 (Fig. 2E). These substrate-binding pocket residues are conserved in Rv0097 and MmaE, but not in ScoE (Supplementary Fig. 7). With CADA bound, the F102 residue is now only in one conformation (F102), creating space to accommodate CADA’s long alkyl chain (Fig. 2E, right, Supplementary Fig. 8). Differential positioning of two loops (pocket loops 1 and 2, Supplementary Fig. 9) in Rv0097 and ScoE additionally create more space for the long alkyl chain of CADA. These structural observations suggest that substitutions of G204 to residues with bulky side chains, such as Phe, would inevitably lead to substantial steric clashes in the presence of the CADA, consistent with a previous report showing that G204F variant dramatically compromises enzymatic activity (as measured by product formation) and perturbs substrate specificity of Rv009712. To examine the structural effect of a G204 substitution, we obtained crystallographic snapshots of a G204F variant without substrate (structure 3 in Supplementary Table 1-2), in complex with the longer CADA substrate (structure 4 in Supplementary Table 1-2), and in complex with the shorter CABA substrate (structure 5 in Supplementary Table 1-2). In the absence of substrate (structure 3), the distance between the G204F and F102 side chains is ~3.8 Å, too close to allow CADA’s 10-carbon alkyl chain to bind in between the two residues (Fig. 2F). Overlay of structures 3 and 2 shows steric clashes between G204F side chain and C8, C9, C10 of the CADA substrate molecule (Fig. 2G), supporting our previous data showing ScoE’s strong preference for short-chain alkyl chain substrates (C4) and Rv0097’s strong preference for long-chain alkyl substrates (>C10)12.

Consistently, structure 4 shows weak electron density for CADA in the presence of the G204F substitution (Fig. 2H). Density is present for the polar end of the substrate, but not for the alkyl chain at its typical binding site. Instead, curved density above G204F is observed in the omit map into which we modeled the alkyl chain of the CADA (Fig. 2H). This curled-up binding pose of CADA is likely to have a considerable steric strain associated with it, explaining why CADA is not present at full occupancy in the structure. The occupancy refined to 0.54 when the B-factors of CADA were reset to match the B-factors of neighboring residues. Similarly, structure 5 shows poor electron density of the shorter-chain CABA, consistent with its weak binding to the active site, with refined occupancy of 0.65 (Fig. 2I).

We additionally obtained kinetics data of the G204F variant for both CADA and CABA (Supplementary Fig. 10). As with the structural studies, we used a racemic mixture of CADA in all our assays. Since we don’t see evidence of (S)-CADA binding in our structures, we believe that the impact of the racemic CADA mixture on enzyme activity is minor, consistent a with previous report26. However, the use of a racemic mixture of CADA and enantiomeric pure CABA is a caveat that should be kept in mind. We find that the enzyme kinetic parameters (kcat and KM) and the catalytic efficiencies (kcat/KM) are similar for both substrates with the G204F variant. The kcat values are similar between the G204F and wild-type enzyme (WT), indicating that when substrate is saturated, the G204F variant catalyzes isonitrile formation at the same rate as WT. However, the KM for the G204F variant against both substrates are much higher than that of WT (kinetics parameters for WT enzyme are from our previous work12), indicating weakened binding affinity of G204F towards both substrates. Due to the higher KM values, the catalytic efficiencies of the G204F variant on both substrates are decreased compared to WT; less than or equal to 30% of the WT enzyme’s catalytic efficiency on CADA (Supplementary Fig. 10).

To delve further into the roles of the two conformations of F102 in gating substrate binding, we prepared three F102 variants: F102W, F102Y, and F102A. We obtained a series of structures of these variants in apo form, in complex with CADA and a vanadyl ion, an Fe(IV)-oxo mimic30–32 (structures 6-10 in Supplementary Table 1-2), or in complex with a vanadyl ion alone (structures 11 in Supplementary Table 1-2). First, we changed F102 to a similar but bulkier aromatic residue Trp (F102W). Notably, in its apo structure at 1.25-Å resolution (structure 6 in Supplementary Table 1-2), the residue W102 is also present in two rotamer conformations (Fig. 3A). The F102W rotamer is more dominant (occupancy = 0.63) than the F102W’ rotamer (occupancy = 0.37). An acetate ion and water molecules are found at the polar end of the CADA binding site. The non-polar end of the CADA binding pocket is still hydrophobic, largely due to the two rotamer conformations of F102W, which seem to prevent water molecules from residing in the cavity. Only one water molecule can be found hydrogen bonded to the nitrogen of F102W’s indole ring. Like the WT enzyme, the distance between the F102W’ rotamer (nitrogen of indole ring) and G204 alpha carbon is short (~4.3 Å) (Fig. 3A). An additional entry point for solvent into the CADA alkyl chain binding pocket is found at residue T203. In structure 6, this entry route is closed due to the side chain of T203 adopting a conformation that allows for a hydrogen bond to the backbone carbonyl carbon of W96 (Fig. 3A), which seals off access to bulk solvent. When CADA is bound, our 2.00-Å resolution structure (structure 7 in Supplementary Table 1-2) shows that only one of the two conformations of F102W is observed, like that in WT (Fig. 3B). However, the single conformation of F102W that is observed is rotated by 180° to avoid steric clashes with the carboxylate of the glycyl moiety of CADA (Fig. 3B). This rotation creates an additional hydrogen bond between the indole nitrogen of F102W and the carboxylate oxygen of CADA (Fig. 3B, magenta dashed line). Overall, when CADA is bound, the substrate-binding site is hydrophobic; water molecules cannot access the pocket at the F102 position or at the T203 position.

We then prepared the F102Y variant and determined its apo crystal structure to 1.37-Å resolution (structure 8 in Supplementary Table 1-2). With an added hydroxyl group in the side chain, F102Y is locked in one conformation by a new 3.2-Å hydrogen bond formed between the hydroxyl groups of Y102 and Y65 (Fig. 3C). Water molecules occupy the site left open by the lack of a second F102Y side chain conformer, filling with solvent molecules the otherwise hydrophobic binding pocket for the alkyl chain of substrate (Fig. 3C). This observation supports a role for F102 in gating access to the substrate-binding pocket. This structure (8) additionally shows that the solvent entry point at T203 is open. The side chain of T203 adopts a different rotamer conformation that creates an opening between the pocket and bulk solvent (Fig. 3C). This alternative T203 side chain conformation is stabilized by a hydrogen bond between the T203 side chain hydroxyl group and a water molecule, which in turn has a hydrogen bond with W96’s backbone carbonyl oxygen (Fig. 3C). When excess CADA is added to the protein for co-crystallization, we find in the resulting 1.46-Å resolution structure (structure 9 in Supplementary Table 1-2) that CADA has displaced the water molecules and that T203 is now sampling both rotamer conformations at approximately equal occupancy (0.54 open to 0.46 closed) (Fig. 3D). Thus, CADA can still bind to the pocket in the F102Y variant, and that binding influences the position of T203.

For the F102A variant, the crystal structure of the apo form at 1.73-Å resolution (structure 10 in Supplementary Table 1-2) shows water molecules taking the place of the bulky side chains of F102 in WT and F102W/F102Y in the variants (Fig. 3E). T203 is in the rotamer conformation that generates the opening to bulk solvent (Fig. 3E). For this variant, even 5-fold excess CADA in the crystallization does not lead to substantial density for CADA’s alkyl chain. The crystal structure at 1.82-Å resolution (structure 11 in Supplementary Table 1-2) shows the side chain of T203 in the open conformation, water molecules in the substrate-binding site, and no significant density for CADA (Fig. 3F). When the difference map is contoured to +2.8 σ, a continuous piece of positive density appears that is reminiscent of the alkyl chain of CADA. However, there is no corresponding density observable in the omit map, hence we did not model CADA into this active site.

To validate the importance of F102, we turned to enzyme activity assays (see Methods). First, both the F102W variant and the F102A variant share slightly lower kcat values compared to the WT enzyme (Table 1, Supplementary Fig. 11), indicating that in substrate-saturating conditions, all three enzymes perform catalysis at comparable rates. However, these three enzymes differ in KM values. The F102W variant has a slightly higher KM value for CADA than WT whereas the F102A variant has a much higher KM value for CADA (in mM range) than WT. Due to these differential KM values, the catalytic efficiency (kcat/KM) of the F102A variant is less than one half of F102W and less than one third of WT, which is the most catalytically efficient (Table 1). In a product formation assay for WT and the three F102 variants, which was conducted with a 10-min incubation period using 1 mM CADA, F102W produced the same amount of product as WT within the error of the experiment, whereas F102A and F102Y variants showed substantially decreased product formation (Fig. 3G). All of these data are consistent with our structural observation that F102W’s substrate-binding site is substantially more hydrophobic with far fewer water molecules for CADA to displace (Fig. 3A) than that of F102A (Fig. 3C) and F102Y (Fig. 3E), hence better suited for binding of the hydrophobic substrate, CADA33,34.

Rv0097 has both open and closed conformations that provide access to the active site
Our co-crystallization and soaking experiments with Fe(II) and/or Fe(IV)-oxo mimic vanadyl (aka oxovanadium(IV) cation)30–32, with CADA and/or αKG, of the WT and variant protein have led to multiple crystal forms (Supplementary Table 1-2). These different crystal forms have allowed us to capture many conformational states of Rv0097. Some of the conformational rearrangements that we have observed are on the smaller side like the rearrangements of the side chains of T203 and of F102 mentioned above. Others are more dramatic and involve substantial loop movements and secondary structure changes (Fig. 4A-B). Fortunately, the Rv0097 crystal lattice turned out to be remarkably malleable, allowing us to obtain crystallographic snapshots of numerous conformational states. Although the soaking of our anaerobically grown Fe(II)-Rv0097 co-crystals in crystallization solutions supplemented with 5 mM CADA for 1 h caused crystals to crack (Supplementary Fig. 12), these damaged crystals still diffracted to high resolution and a 1.74-Å resolution structure was obtained (structure 12 in Supplementary Table 1-2). Indexing of data indicated the same space group (P21) but an expanded unit cell with four protomers in the ASU, instead of two, and a solvent content of ~42%. Close examination of packing in the crystal lattice reveals that the new ASU contains a homodimer like in previous structures (Supplementary Fig. 13A-C) and two additional protomers each from a different homodimer (Supplementary Fig. 13D, E). Since the space group remain the same, this new ASU with four chains is related to another ASU along the b direction by a screw axis (Supplementary Fig. 13F).

Although CADA is not bound in any of the structure 12 protomers, this soaking experiment led to a structure with a wide-open active site (open state) and to three structures in which the active site is closed (closed state) (Fig. 4A). Overlayed structures of open (pink in Fig. 4A) and closed states (yellow in Fig. 4A) (all atom RMSD = 2.295 Å, Cα RMSD = 2.189 Å) reveal significant conformational rearrangements of the dimer interface region (“left lid”, residues D152 to P181) and a region on top of the active site (“right lid”, residues Y65 to F95) (Fig. 4A). The movement of the “right lid” affects the positioning of two surface loops that are important for αKG positioning; the His loop and Arg loop, which are discussed in the following section. The movement of the “left lid” involves a 18.6° rotation to open the active site (Fig. 4A, green angle). The first half of the “right lid”, residues Y65 to I78, undergoes an upward ~2.5 Å shift (Fig. 4B, green arrow), whereas the second half, I78 to F95, undergo more drastic and complex conformational rearrangement that involve changes in protein secondary structures (Fig. 4B). In the closed state (yellow in Fig. 4), residues I78-S82 form a short β-strand (strand 1) that hydrogen bonds to an adjacent β-strand (strand 2), followed by a long-ordered loop (residues S82-F95). In the open state (pink in Fig. 4), residues S82-E85 undergo a loop-to-strand secondary structure change, and the tail of strand 1 is extended from S82 to E85. Strand 2 is also extended with residues G269-T272 undergoing a similar loop-to-strand rearrangement that results in strand 2’s N-terminus starting at residue G269 instead of at T272. The extended parts of both strands are held together by backbone hydrogen bonds. Residues E85-G93 in the open state are disordered, thus exposing the active site to the bulk solvent (Fig. 4C). A pool of ordered water molecules occupies the CADA-binding site and the site near R122/H264 where αKG is expected to bind (Fig. 4C). Both R122 and H264 are pointing toward the putative αKG binding site even though αKG is not present (Fig. 4C). In contrast, the active site in the closed state has far fewer solvent molecules (Supplementary Fig. 14A). Our observed open and closed protein conformational states have been seen before in the family of mononuclear non-heme iron enzymes. In the prototypical non-heme Fe(II)/αKG-dependent dioxygenase TauD, substrate taurine binding induces a conformational change of TauD from open to closed35 (Supplementary Fig. 15). Like Rv0097, a “left lid” and a “right lid” could be identified that undergo structural rearrangements as substrate binds in the active site (Supplementary Fig. 15). However, no secondary structure change is observed.

Since soaking of anaerobically grown Fe(II)-Rv0097 co-crystals with 5 mM CADA did not yield substrate density, we doubled the soaking concentration of CADA to 10 mM and solved the structure to 1.76-Å resolution (structure 13 in Supplementary Table 1-2). Again, this crystal form contained four chains in the ASU, one open state structure and three closed state structures. In all three of the closed state structures, clear density corresponding to CADA molecules, and not acetate molecules, were now present (Supplementary Fig. 14B, C). To obtain a structure with both CADA and αKG bound, we soaked anaerobically grown Fe(II)-Rv0097 co-crystals with 5 mM of both CADA and αKG. We obtained a 1.82-Å resolution structure (structure 14 in Supplementary Table 1-2) in which we observed strong electron density that corresponds to an αKG bound bidentate to Fe(II) (Fig. 4D). However, no density was observed for CADA. We find that αKG is bound to Rv0097 with its C5 carboxylate positioned to form a salt bridge with the side chain of R122 (Fig. 4D). Based on the ScoE structure with tartrate, we proposed that R122 of Rv0097 and the equivalent ScoE arginine (R157) would be responsible for anchoring αKG in the active site. Now we know that for Rv0097, our proposal is correct. However, this αKG is bound to the Fe site in what appears to be an offline orientation with the Fe-binding C1 carboxylate oxygen occupying the expected position of the putative Fe(IV)-oxo intermediate. αKG is said to be offline when its C1 carboxylate oxygen is trans to the second His in the His…Asp(Glu)…His triad, requiring that the Fe(IV)-oxo is cis to that second His36 (Supplementary Fig. 16). With αKG bound in this manner, the only open coordination site on the Fe(II) ion is facing away from the substrate binding site, meaning that the Fe(IV)-oxo intermediate formed by the molecular oxygen-dependent cleavage of αKG would not be positioned correctly for substrate hydrogen atom abstraction. Thus, we expect that either αKG rearranges upon substrate or molecular oxygen binding or that the Fe(IV)-oxo intermediate flips closer to substrate following the αKG cleavage reaction. Both αKG rearrangements37 and Fe(IV)-oxo intermediate flips38 have been proposed in the mechanisms of other Fe(II)/αKG-dependent dioxygenases.

In a further attempt to obtain a Fe(II)•αKG•CADA•Rv0097 ternary complex structure, we co-crystallized Fe(II), CADA and Rv0097, and then soaked these crystals in 10 mM αKG. The resulting structure at 1.45-Å resolution (structure 15 in Supplementary Table 1-2) excitingly shows density for both CADA and αKG (Fig. 4E). We find both R122 and H264 pointing toward the Fe center and that αKG is bound to Fe(II) in the same putative offline orientation, as described above, again making the salt bridge interactions between C5 carboxylate of αKG and R122 side chain. CADA binds to the substrate-binding site with no steric clashes with αKG, indicating that this offline αKG conformation can accommodate substrate in the active site (Fig. 4E). Structure 15 suggests that it is not substrate binding that leads αKG to adopt an online conformation (see Supplementary Fig. 16). However, since the αKG was soaked rather than co-crystallized, it is possible that the enzyme was not able to undergo the conformational change required for an online αKG conformation, and that in solution, substrate binding does cause αKG to adopt an online conformation. Alternatively, as mentioned above, it is also possible that molecular oxygen binding to Fe(II) triggers a conformational change in αKG or that this conformation is in fact online, and that it is the Fe(IV)-oxo intermediate that rearranges. More work is needed to resolve these specific questions.

Conformational changes associated with open and closed states affect CADA-gating residues
The conformational changes associated with the open and closed enzyme structural states directly affect substrate access to the active site and substrate-binding residues (Fig. 4F). Notably, in the open conformation, F102 adopts the F102’ conformation and point towards G204. Surface representation of the open chain suggests that the active site is accessible for substrate binding (Fig. 4G, circled region). F102’ directly stacks against a neighboring P153 residue and is stabilized by C-H(P153) ⋯π(F102’) interaction, with centroid-to-centroid distance of 4.1 Å (Fig. 4H). Such C-H⋯π interactions between an aromatic residue and a Pro residue has precedence in protein structures39. For example, in the glycyl radical enzyme HypD, the substrate trans-4-hydroxy-L-proline is stabilized by a proline-aromatic interaction with F340 residue40. Sequence and structural alignments of Rv0097 with ScoE indicate that this P153 residue is not conserved. In ScoE, V188 takes the place of P153, and is similarly stacked on top of F102’s counterpart F137, with a distance of 3.7 Å (Supplementary Fig. 17). To test the importance of P153 in Rv0097, we prepared two enzyme variants of this P153 residue, P153V and P153A. Activity assay monitoring product formation on both variants showed decreased activity (Fig. 4I), suggesting that weakening this C-H⋯π interaction probably destabilized the open conformation of the enzyme, thereby affecting substrate binding, hence leading to lower enzymatic activity.

Movements of a His loop and Arg loop provide a different route into the active site for second αKG
Our previous work on ScoE13 showed that both H299 and R157 are important for catalysis. Here we show the same is true for Rv0097’s H264 and R122 (Fig. 5A). Activity assays monitoring product formation on H264/R122 variants show that both residues are critical for enzymatic activity (Fig. 5B), with R122 being the more important one, likely because of its essential role in αKG coordination. Based on the ScoE structure with tartrate bound, we proposed an inducible αKG-binding mechanism for ScoE in which loops containing H299 and R157 undergo large conformational changes to create a binding site for αKG every time that αKG binds. Arg is a common αKG-binding residue, but in other Fe(II)/αKG-dependent dioxygenases, the Arg does not move and the αKG binding site is not inducible17. We speculated a few reasons why ScoE might have evolved an inducible mechanism for αKG binding, including that such a mechanism would aid in binding specificity for two co-substrates (CABA and αKG) that are structurally similar to each other. Given that CADA is less similar to αKG due to CADA’s long alkyl chain, CADA would be less likely to compete with αKG for binding to the same site, potentially negating the need for an inducible αKG-binding site. In this work, we find that Rv0097 does have an inducible αKG-binding site. We observe different conformations of the loops (the His loop and the Arg loop), which contain the analogous residues to ScoE’s H299 and R157; Rv0097’s H264 and R122 (Fig. 5A).

Overlaying the WT structures (1, 2, 12, 15, 16) and ScoE structures (PDB: 6XO313, 6L6X15) reveals different conformations for H264/H299 and R122/R157 side chains, which can be generally classified as inward (toward active site) and outward (away from active site) (Fig. 5C). Interestingly, a 1.36-Å resolution vanadyl and CADA bound structure (structure 16 in Supplementary Table 1-2) shows additional movement of H264 further towards the active site in a position analogous to that of H299 of ScoE with tartrate bound (Fig. 5C). Like ScoE, CADA binding to Rv0097 did not promote a conformational change in the His/Arg residues: both apo (structure 1, Supplementary Fig. 18) and the Fe(II)- and CADA-bound structure (2) have R122 and H264 residues in the outward conformation. However, when Rv0097 is in the open state (open state in structure 12, 13, 14) or when both substrates (CADA and αKG) are bound (structure 15), R122 swings towards the active site and is in an inward conformation, ready for αKG binding. The positioning of R122 is similar to that of R157 of ScoE when tartrate is bound (Fig. 5C).

The structural overlay of the WT Rv0097 structures 1, 2, 14, 15, 16 revealed three distinct His loop conformations (H1, H2, and H3) and two distinct Arg loop conformations (R1 and R2), as represented by the three structures shown in Fig. 5D. The H1 and H2 conformations of the His loop and both Arg loop conformations are observed in two chains of the apo structure (Supplementary Fig. 18). Closer inspections of H264/R122 side chain positions in these structures and their corresponding loop conformations reveal correlated motions. When the His loop is in H1 conformation (nearest to the Arg loop), the Arg loop is in the R1 conformation (Fig. 5D, left). R122 side chain is in outward conformation to form a 2.7 Å hydrogen bond with backbone carbonyl oxygen of A267 (Fig. 5D, right). When the His loop moves to the left (H1 to H2), the Arg loop moves to the left with it (R1 to R2). For the Arg loop, going from R1 to R2 involves backbone peptide flips of P118 and G119 (Fig. 5D). As the side chain of R122 moves outward, the His loop moves accordingly to prevent the steric clashes between R122 side chain and backbone carbonyl of A267 (Fig. 5D).

Interestingly, we observe two discrete His loop conformations in the same chain of our vanadyl and CADA bound structure (structure 16). In addition to showing a H1 conformation, we find a H3 conformation not previously observed in other structures (Fig. 5D). The Arg loop is also in two discrete conformations (R1 and R2) that differ primarily by backbone flips at P118 and G119 (Fig. 5D). Refined occupancies of the loops suggest that there is a major loop pair H3/R2 (~0.65 occupancy, olive green in Fig. 5E) and a minor loop pair H1/R1 (~0.35 occupancy, light green in Fig. 5E). In the major H3/R2 pair, H264 is pointing inward and R122 outward, and in the minor pair, H264 has flipped and is now pointing outward whereas R122 has changed positions but is still outward.

The His loop is the extension of strand 2 that is part of the “right lid,” which undergoes conformational changes so that CADA can bind, and products can leave. As the “lids” open, the Arg loop moves from R2 to R1 conformation, and the His loop moves from H2 to H1 position and R122 becomes positioned for αKG binding (Fig. 5F, left). The series of loop movements is a result of the loop-to-strand secondary structure change of strand 1 in the open state. Strand 1 in the open state (pink in Fig. 5F, right) would have steric clashes with His loop when it is in H2 conformation (1.2 Å distance between E85 of strand 1 in open state and G269 of His loop in H2 conformation) (Fig. 5F, right); therefore, H2 to H1 conformational change is necessary. Arg loop thus moves accordingly from R2 to R1 conformation.

In the vanadyl and CADA bound structure 16, the conformation of the major H3/R2 pair creates a space in between the two loops large enough for the long side chain of R122 to swing out to its outward conformation without clashes with His loop backbone (Supplementary Fig. 19). We hypothesize that this opening could also serve as an access channel for the dissociation of αKG-reaction products, succinate and CO2 following the first half reaction, and the association of a second αKG for the second half-reaction. R122 side chain being in the most extended outward conformation makes it easier to bring αKG into the Fe site without having to fully open the enzyme (left and right “lids” in open conformation) during the catalytic cycle, thereby keeping CADA-intermediates protected. When the enzyme is in the open state, His/Arg loops are locked in position (the H1/R1 conformation), so they are unable to form the H3/R2 access channel, but this access channel is not necessary when the enzyme is in the open state. When open, the active site is readily accessible for co-substrate entry and co-product exit as is required at the start of turnover and at the end of the second half reaction when turnover is complete.

H264 seems to play a role in securing the various loop conformations so that αKG and its cleavage products can come and go in between the two half reactions without opening up the active site to solvent, protecting the CADA-intermediate. When αKG is bound to an inward pointing R122 (structure 15), the H264 side chain hydrogen bonds to the backbone of the adjacent loop (backbone of T92) via an intermediary water molecule that is close to the active site (Fig. 5G), stabilizing the loop conformations and preventing solvent from entering the active site. When succinate and CO2 leave and R122 points outward (major loop pair in structure 16), as is expected in between two half-reactions, the H264 side chain directly forms a 3.3 Å hydrogen bond with T92 backbone carbonyl, further sealing up the reaction site and protecting reactive intermediate species Fig. 5G). H264 can only be in this most inward position when R122 points outward, since the two residues would sterically clash if both were pointed inward (green H264 and red R122 in Fig. 5G). Overall, these observed conformational changes of H264/R122 side chains and the His and Arg loops are similar to what was reported for ScoE (Figs. 5G and 5H), but now we know that these changes are related to αKG binding and are not an artifact of tartrate in the ScoE structure.

Imine-CADA intermediate appear to stay bound to enzyme between half reactions
To investigate whether the imine-CADA intermediate remains sequestered within the enzyme active site between two half-reactions, we monitored imine-CADA formation in Rv0097-catalyzed reactions employing our previously established protocol14. Here we derivatized an acid-catalyzed degradation product of imine-CADA, glyoxylate, using 2-nitrophenylhydrazine (2-NPH)14. Our assays monitor imine-CADA production under three conditions: (1) full reaction condition, where the complete enzymatic reaction mixture was directly analyzed; (2) filtrate condition, where the enzyme was removed by centrifugation and only the filtrate was analyzed; and a no-enzyme control. After 1-minute incubation periods, we observed significantly higher glyoxylate production in the full reaction condition (Supplementary Fig. 20), suggesting that the imine-CADA intermediate stay bound to the enzyme during catalysis.

Methods
Commercial materials
Q5 High-Fidelity PCR Master Mix (New England Biolabs) was used for polymerase chain reactions (PCR). Restriction enzymes were purchased from New England Biolabs. Oligonucleotides were purchased from ELIM BIOPHARM. All chemicals used in this work were purchased from Sigma-Aldrich, Fisher Scientific, Alfa Aesar, or Hampton Research, unless otherwise noted. 3-aminodecanoic acid hydrochloride (purity: 95%) was purchased from Enamine.

Construction of plasmids for expression in E. coli
The rv0097 gene was PCR amplified from genomic DNA (Mycobacterium tuberculosis H37Rv) and cloned into pET24b+ through restriction enzyme digestion (ThermoFisher) and ligation with NEBuilder HiFi DNA Assembly (New England Biolabs). The primers and vectors used in this study are reported in Supplementary Table 3 and 4, respectively. Plasmids were extracted using a Zyppy Miniprep Kit (Zymo Research) and confirmed by DNA sequencing at ELIM BIOPHARM.

Site directed mutagenesis
Rv0097 variants were constructed using the Agilent QuickChange II Site Directed Mutagenesis kit by following the manufacturer’s protocol with the primers listed in Supplementary Table 3. pET24b+-Rv0097 was utilized as a template for the PCR reactions. The introduction of point mutations was confirmed with DNA sequencing and the corresponding Benchling link to the sequencing data are listed in Supplementary Table 4. E. coli. strains used are listed in Supplementary Table 5.

Protein expression and purification for assays
The expression and purification of Rv0097 was conducted using a similar procedure as previously reported12,45. BL21 Star (DE3) competent cells were inoculated (2% inoculum ratio) to 1 L of Terrific Broth (TB) in a shake flask containing 50 μg/mL of kanamycin. The cultures were grown at 37 ⁰C at 250 RPM to an OD600 of 0.6. The cells were then cooled on ice for 10 min and induced with 250 μM isopropyl-β-D-galactopyranoside (IPTG) for 16 h at 16 ⁰C and 250 RPM. Cells were harvested by centrifugation (6,371 xg, 15 mins, 4 ⁰C), resuspended in 30 mL of lysis buffer (50 mM HEPES pH 8.0, 500 mM NaCl, 5 mM imidazole), and lysed by sonication on ice. Cellular debris was removed by centrifugation (27,216 xg, 45 min, 4 ⁰C), and the supernatant was filtered with a 0.45 μm filter before batch binding. Nickel-Nitrilotriacetic acid (Ni-NTA) resin (Qiagen) was added to the filtrate at 3 mL/L of culture, followed by incubation for 1 h at 4 ⁰C. The protein-resin mixture was loaded onto a gravity flow column in which the flow through was discarded. The column was then washed with approximately 30 mL of wash buffer (50 mM HEPES pH 8.0, 100 mM NaCl, 20 mM imidazole). Proteins were eluted in approximately 15 mL of elution buffer (50 mM HEPES pH 8.0, 100 mM NaCl, 250 mM imidazole). The entire process was monitored with a Bradford assay. Rv0097 was concentrated to 10 mL using a 10 kDa Amicon spin filter and subsequently dialyzed using a 10 kDa Slide-A-Lyzer cassette overnight in 2 L of dialysis buffer (50 mM HEPES pH 8.0, 100 mM NaCl, 1 mM EDTA). The dialysis buffer was changed twice during the overnight period (every 2 h). Rv0097 was concentrated using a 10 kDa Amicon spin filter to 40 mg/mL (with 10% v/v glycerol). Rv0097 was flash frozen in liquid nitrogen and stored in 30 μL beads at −80 ⁰C. The presence and purity of purified proteins were assessed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was determined by UV/Vis absorbance at 280 nm using an extinction coefficient of 45,380 M−1 cm−1 that was determined using the ProtParam tool46.

The approximate molecular weight and protein yield of Rv0097 are as follows: 34 kDa, 10 mg/L from TB, Uniprot: P9WG83. The Rv0097 variants had purification yields that were nearly the same as that of the WT enzyme (±8%).

Reconstitution of apo-Rv0097 to yield holo-Rv0097 for assays
A Bio-Rad Bio-Gel P-6 gel column was equilibrated in exchange buffer (50 mM HEPES pH 8.0, 100 mM NaCl). Rv0097 was desalted to remove EDTA by following the manufacturer’s protocol. The enzyme was immediately added to the biochemical assay and supplemented with ammonium iron(II) sulfate hexahydrate matching 90% of the protein concentration.

Synthesis of CADA and CABA
In general, the syntheses of racemic CADA and enantiomerically pure CABA were followed as previously reported12,45. CADA was isolated as a colorless oily liquid and confirmed by high-resolution mass spectrum (HRMS) and nuclear magnetic resonance (NMR). NMR spectra were recorded with a Bruker AVANCE spectrometer at 900 MHz (1H NMR), and 226 MHz (13C NMR). NMR solvents were purchased from Cambridge Isotope Laboratories Inc. Other chemicals and solvents were purchased from Sigma-Aldrich. High-performance liquid chromatography (HPLC) purification was carried out using an Agilent 1200 system and a Grace Alltima C18 column (150 × 10 mm). LC-HRMS was conducted using an Agilent 6545 Accurate-Mass Q-TOF. CADA: calculated for C12H23NO4 [M+H]+: 246.1700; found 246.1695 (2 ppm error). 1H NMR (900 MHz, DMSO-d6) δ 3.86 (s, 2H), 3.47 (m, 1H), 2.75 (dd, J = 17.1, 5.1 Hz, 1H), 2.59 (dd, J = 17.1, 6.8 Hz, 1H), 1.71 (m, 1H), 1.55 (m, 1H), 1.30–1.20 (m, 10H), 0.86 (t, J = 7.1 Hz, 3H). 13C NMR (226 MHz, DMSO-d6) δ 171.96, 168.34, 54.12, 45.06, 34.83, 31.12, 30.48, 28.67, 28.39, 24.37, 22.06, 13.94. CABA: calculated for C6H12NO4 [M+H]+: 162.0761; found 162.0762 (0.6 ppm error). 1H NMR (900 MHz, DMSO-d6): δ 3.91 (d 144.0, 2H), 3.54 (m, 1H), 2.84 (dd 16.6, 4.3, 1H), 2.50 (m, 1H), 1.25 (d 6.5, 3H). 13C NMR (225 MHz, DMSO-d6): δ 171.6, 168.4, 50.3, 44.8, 37.1, 16.2. Mestrenova version 9.0.1 was used to analyze NMR data. LC-HRMS data were analyzed using Agilent MassHunter Qualitative Analysis software 10.0.

Activity assays and quantification of isonitrile
A 100 μL biochemical assay consisting of 50 mM HEPES pH 8.0, 1 mM CADA, 2 mM αKG, 100 μM apo-Rv0097, and 90 μM Fe(NH4)2(SO4)2 was incubated at room temperature for 10 min. The enzymatic reaction was quenched with 200 μL of 667 μM 3,6-di(pyridine-2-yl)-1,2,4,5-tetrazine (Py-tetrazine) dissolved in cold methanol, gently mixed, and incubated for 1 h at room temperature. The quenched reaction mixture was further vortexed and centrifuged for 10 min to remove aggregated protein. LC-HRMS analysis was conducted on an Agilent Technologies 6545 Accurate-Mass QTOF LC-MS instrument and an Eclipse Plus C18 column (100 × 4.6 mm). Chromatography was performed using a linear gradient of 10-50% acetonitrile (v/v) with 0.1% formic acid over 12 min in water with 0.1% formic acid at a flow rate 0.5 mL/min. The production of INDA was estimated by looking for the formation of 3,5-di(pyridine-2-yl)-1Hpyrazol-4-amine (Py-AP) and comparing its retention time and mass spectrum with a standard. The Py-AP standard was synthesized and characterized by NMR47 was observed with [M+H]+: 238.1088 (calculated [M+H]+: 238.1086, 0.8 ppm error). Negative controls omitting CADA, αKG, and Rv0097 were also conducted. LC-HRMS data were analyzed using Agilent MassHunter Qualitative Analysis software 10.0.

Determination of kinetic parameters of Rv0097 towards CADA
100 μL biochemical assays were performed in triplicate containing 50 mM HEPES pH 8.0, 5 mM αKG, 10 μM apo-Rv0097, and 9 μM Fe(NH4)2(SO4)2. The reactions were initiated by adding αKG and CADA was supplied at varying concentrations depending on the Rv0097 variant in question. After incubation, the enzymatic reaction was quenched with 200 μL of 667 μM Py-tetrazine dissolved in cold methanol, gently mixed, and incubated for 1 h at room temperature. The subsequent reactions were centrifuged to remove protein debris, and the supernatant was analyzed with LC-HRMS. Time points were taken at 30 s, 1 min, 2 min, 5 min, and 10 min to determine the initial velocity of INDA formation. The product concentration was estimated by constructing a standard curve of Py-AP by analysis of standards containing 100 μL of 50 mM HEPES pH 8.0 and varying amounts of Py-AP that were subsequently quenched with 200 μL of 667 μM Py-tetrazine dissolved in cold methanol. Chromatography was performed using a linear gradient of 10-50% acetonitrile (v/v) with 0.1% formic acid over 12 min in water with 0.1% formic acid at a flow rate 0.5 mL/min. Kinetic parameters were then calculated using GraphPad Prism 9. LC-HRMS data were analyzed using Agilent MassHunter Qualitative Analysis software 10.0.

Estimation of Rv0097-bound imine-CADA versus free
A 200 μL assay containing 50 mM HEPES pH 8.0, 1 mM CADA, 1 mM αKG, 50 μM apo-Rv0097, and 45 μM Fe(NH4)2(SO4)2. The reaction was split into two and performed as described14. Briefly, reactions were quenched after short incubation periods (1 min), followed by chemical derivatization to assess glyoxylate formation for imine intermediate quantification. To distinguish between free and enzyme-bound imine, the relative production from the reaction supernatant alone (enzyme is removed via filtration) to that of the complete reaction mixture containing the enzyme. A no enzyme control was also performed. Results are shown in Supplementary Fig. 20).

Protein expression and purification for crystallization
To aid soluble expression and maximize protein yield, Rv0097 WT and its G204F, F102Y, F102W, and F102A variants were co-expressed with molecular chaperones using an auto-induction protocol, as follows. 1.0 μL of 10 ng/μL pET24b+-Rv0097 plasmid (see above for plasmid construction) was mixed with 1.0 μL of 10 ng/μL chaperone plasmid pGro748 (Takara Bio) that contains groES-groEL under araB promoter and chloramphenicol antibiotic resistant marker. The resulting 2.0 μL plasmid mixture was co-transformed into BL21 Star (DE3) Chemically Competent E. coli (Invitrogen, ThermoFisher). A single colony was picked to inoculate an overnight starter culture (16 hrs) in 50 mL LB medium containing 50 μg/mL kanamycin (GoldBio) and 25 μg/mL chloramphenicol (OmniPur, EMScience), with 220 RPM shaking at 37 ⁰C. 5 mL of the overnight starter culture was used to inoculate 1 L of autoclaved auto-induction media (prepared following Studier, F. W49.) containing 150 μg/mL of kanamycin and 25 μg/mL chloramphenicol. 2.0 g of L-(+)-arabinose (Sigma-Aldrich) was added to the 1 L culture during inoculation to allow for chaperone expression prior to Rv0097 expression. Cells were grown with 220 RPM shaking at 37 ⁰C until OD600 reached ~0.6. Cell cultures were taken out of the incubator and cooled at 4 ⁰C for 30 min without shaking, after which they were put back to the incubator for an additional 20 h with 220 RPM shaking at 22 ⁰C for protein expression. Cells were harvested by centrifugation (9000 xg, 10 mins, 4 ⁰C), and flash-frozen in liquid nitrogen before storage at −80 ⁰C until cell lysis and protein purification.

Cells from 1 L culture were taken out of −80 ⁰C and let thaw under 4 ⁰C, then resuspended in 50 mL of lysis buffer (50 mM HEPES pH 8.0, and 500 mM NaCl) supplemented with 1 mg/mL egg-white lysozyme (GoldBio), 0.15 μL/mL benzonase nuclease (Sigma-Aldrich), and 1 tablet of cOmplete EDTA-free protease inhibitor cocktail (Sigma-Aldrich), and put on a nutator for 1 h at 4 ⁰C to allow complete thawing and resuspension. Cell lysis was completed by ultrasonication on ice using the following parameters: 60% amplitude, 8 cycles of 30 s, 1 s on 2 s off per cycle. The resulting suspension was clarified by centrifugation (28,000 xg, 50 min, 4 ⁰C). The clarified lysate was filtered using 0.45 μm filter before loading onto a 5-mL HiTrap TALON crude column (Cytiva) pre-equilibrated with 10 column volumes (CV) of lysis buffer at 5 mL/min flow rate, followed by 30 min of batch-binding to ensure maximized protein binding. The column was then washed with 25 CV of wash buffer (50 mM HEPES pH 8.0, 300 mM NaCl, and 5 mM imidazole) and eluted with 10 CV of elution buffer (50 mM HEPES pH 8.0, 300 mM NaCl, and 150 mM imidazole). The eluted protein was concentrated using a 10 kDa MWCO (Amicon) centrifugal filter to a final volume of 2.0 mL, then buffer-exchanged by dialysis under 4 ⁰C against 800 mL of dialysis buffer (50 mM HEPES pH 8.0, 300 mM NaCl, 1 mM acid EDTA) using a 10 KDa MWCO Slide-A-Lyzer dialysis cassette (ThermoFisher). The dialysis buffer was changed once after 4 h before overnight dialysis (~18 h). Buffer-exchanged protein was further purified by a 1 CV run of size-exclusion chromatography (SEC) by loading onto a Superdex 200 16/60 size-exclusion column pre-equilibrated with 2 CV of SEC buffer (25 mM HEPES pH 8.0, and 100 mM NaCl). Fractions correspond to Rv0097 dimer were pooled and concentrated using a 10 kDa MWCO (Amicon) centrifugal filter until a final concentration of 10.0 mg/mL (0.3 mM). Throughout purification, protein purity was monitored by 4 to 20% (w/v) SDS-PAGE. C-terminal purification tag (amino acid sequence: KLAAALEHHHHHH) was not cleaved. Protein concentration was determined by UV/Vis absorbance at 280 nm using an extinction coefficient of 45,380 M−1 cm−1 that was determined using the ProtParam tool46. Concentrated protein was either immediately used for crystallization (see below), or aliquoted and flash-frozen in liquid nitrogen then stored at −80 ⁰C for future use.

Crystallization
Rv0097 WT purified as described above was used directly to identify initial crystallization conditions using PEG/Ion HT crystallization screen (Hampton Research) dispensed by an NT8 crystallization robot (Formulatrix) to 96-well sitting-drop trays at room temperature. The identified conditions were further optimized using hanging-drop vapor diffusion method at room temperature. All chemicals used for crystallization were purchased from Hampton Research. Crystals of apo-Rv0097 1 were obtained by mixing 1.0 μL of apo Rv0097 and 1.0 μL of well solution (270 mM magnesium acetate and 16% w/v PEG 3,350), in a sealed well over 500 μL of well solution. Protein crystals typically appeared in 3–5 days and continued to grow over 2-3 weeks. After 2 weeks, crystals were harvested, cryoprotected with paraffin oil, and flash frozen in liquid nitrogen, leading to the apo dataset for Rv0097. Several drops containing crystals of apo-Rv0097 were used to create a seed stock using seed bead kit (Hampton Research). Briefly, drops were combined and vortexed for 3 min total, with one-minute breaks on ice for every 30 s of vortexing. For long-term storage, prepared seed stocks were flash frozen in liquid nitrogen and stored at −80 ⁰C until next use. The seed stock and its serial dilutions (up to 100,000-fold dilution) were used for subsequent micro-seeding crystallizations. To obtain the apo-G204F 3/F102Y 6/F102W 8/F102A 10 structures, aerobically purified protein (10.0 mg/mL, 0.3 mM) was directly used for crystallization using diluted seed stocks. The hanging drops consisted of 1.0 μL of Rv0097 variant and 1.0 μL of diluted seed stock, in a sealed well over 500 μL of well solution. Crystals grew out of micro-seeds typically appeared in 2 days (crystals appeared overnight in drops with more concentrated seed stock) and continued to grow to full size in 1–2 weeks. After 2 weeks, these crystals, and all crystals described below, were harvested, cryoprotected with paraffin oil, and flash frozen in liquid nitrogen.

To obtain Fe-CADA Rv0097 co-crystals 2, frozen aliquots of purified Rv0097 (10.0 mg/mL, 0.3 mM) were brought into an MBraun anaerobic chamber in an N2 environment and let thaw on cold beads. After thawing, the protein aliquots were allowed to equilibrate on cold beads for at least 30 mins with PCR tubes open before adding 1 molar equivalence (0.3 mM) of Fe(NH4)2(SO4)2, 5 molar equivalence (1.5 mM) of CADA (see above for its synthesis), and 5 molar equivalence (1.5 mM) of αKG (α-Ketoglutaric acid disodium salt dihydrate, Sigma-Aldrich) (1.5 mM), then gently mixed and incubated overnight at 4 ⁰C. The hanging drops consisted of 1.0 μL of Fe- and CADA- supplemented Rv0097 and 1.0 μL of diluted seed stock, in a sealed well over 500 μL of well solution. Crystals started to appear in 1–2 days and grew to full size within 2 weeks. These crystals were then transferred to a Coy anaerobic chamber with an Ar/N2 gas mix environment for looping. Crystals were then harvested, cryoprotected with paraffin oil, and flash frozen in liquid nitrogen. To obtain the ternary complex structure 15, the Fe-CADA Rv0097 co-crystals were looped and deposited into a 3.0 μL drop of soaking solution that contains 10 mM αKG and 16% w/v PEG 3,350 for 1 h prior to looping. For anaerobic looping, the soaked crystals were transferred to a Coy anaerobic chamber with an Ar/N2 gas mix environment for cryoprotection and cryo-cooling.

To obtain the CADA- and/or αKG-soaked crystals, frozen aliquots of purified Rv0097 (10.0 mg/mL, 0.3 mM) were brought into an MBraun anaerobic chamber in an N2 environment and let thaw on cold beads. After thawing, the protein aliquots were allowed to equilibrate on cold beads for at least 30 mins with PCR tubes open before adding 1 molar equivalence (0.3 mM) of Fe(NH4)2(SO4)2 then gently mixed and incubated overnight at 4 ⁰C. The hanging drops consisted of 1.0 μL of Fe- and CADA- supplemented Rv0097 and 1.0 μL of diluted seed stock, in a sealed well over 500 μL of well solution. Crystals started to appear in 1-2 days and grew to full size within 2 weeks. Then, the crystals were looped and deposited in 3.0 μL drops of soaking solutions and soaked for 1–2 h over sealed wells prior to looping. The soaking solutions contained the following: (1) 5 mM CADA and 16% w/v PEG 3350 for the acetate-bound open conformation structure (5 mM CADA soaked structure 12); or (2) 10 mM CADA and 16% w/v PEG 3350 for the CADA-bound open conformation structure (10 mM CADA soaked structure 13); or (3) 5 mM CADA, 5 mM αKG, and 16% w/v PEG 3350 for the CADA- and αKG-bound open conformation structure (5 mM CADA and 5 mM αKG soaked structure 14). Crystals were looped and cryo-cooled in a Coy anaerobic chamber using the conditions described above.

The Vanadyl-bound Rv0097 crystals were grown in aerobic conditions. To obtain the vanadyl-CADA WT co-crystals 16, vanadyl-CADA G204F 4/F102Y 9/F102W 7/F102A 11 variants co-crystals, and vanadyl-CABA-G204F 5 co-crystals, aerobically purified protein was directly supplemented with 1.3 molar equivalence (0.4 mM) of Vanadium(IV) oxide sulfate hydrate (Sigma-Aldrich), 5 molar equivalence (1.5 mM) of CADA (or CABA for the vanadyl-CABA-G204F structure 5), and 5 molar equivalence (1.5 mM) of succinate (succinic acid, pH 7.0, Hampton Research), and incubated on ice for 2 h. The resulting protein solution was used for crystallization using diluted seed stocks. Crystals started to appear in 1–2 days and grew to full size within 2 weeks. Crystals were looped and cryo-cooled aerobically using conditions described above.

X-ray data collection and processing
X-ray diffraction data were collected at cryogenic temperature (100 K) at the Stanford Synchrotron Radiation Lightsource (SSRL) (Menlo Park, CA) beamline 9–2 using a Dectris Pilatus 6M detector. All data were indexed, integrated, and scaled in XDS, with data collected at the Vanadium peak wavelength processed anomalously with Friedel pairs kept separate. Resolution cutoffs were determined based on collective considerations of Rsym, CC1/2, I/σ, and data completeness in the highest resolution bin. Data collection statistics are summarized in Supplementary Table 1.

Structure determination and refinement
The Rv0097 WT apo structure 1 was solved by molecular replacement (MR) using the crystal structure of Rv0097 homolog from Streptomyces coeruleorubidus, ScoE13 (45% identity, 61% similarity), as the search model (PDB ID: 6XOJ). Prior to MR, the model was improved in phenix.sculptor50 by gap-based mainchain deletion (sequence alignment-based deletion of residues present in the search model but not in the target) and Schwarzenbacher-style51 sidechain pruning. Edited search model was then used for MR in phenix.Phaser-MR52 using data extended to full resolution. The final MR solution with LLG of 810.6 and TFZ 29.9 identified two protomers in the asymmetric unit (ASU), corresponding to a Rv0097 dimer, consistent with our SEC data that Rv0097 exists as a homodimer in solution. This final MR solution was refined in phenix.refine53 first using rigid-body refinement, with 5% of total reflections randomly selected and set aside for the test set (Rfree). One round of rigid-body refinement led to an initial Rfree of 0.49. Several following rounds of positional refinement and group B-factor refinement were sufficient to reduce Rfree to 0.44. Iterative rounds of positional and individual B-factor refinements were then performed, with manual adjustments in Coot54 against 2mFo-DFc maps contoured to 1.0 σ. Two-fold non-crystallographic symmetry (NCS) restraints were used throughout the refinements. A 2mFo-DFc simulated-annealing composite omit map was generated with 5% of the model omitted for individual omit maps, using Phenix Composite_omit_map55 after each round of refinement to verify this structure and all structures described below. Water and other solvent molecules from crystallization conditions were manually added to this structure, and to all structures described below, using mFo-DFc map contoured to 3.0 σ and the composite omit map contoured to 1.0 σ as criteria. All 2mFo-DFc and mFo-DFc maps are generated by phenix.refine.

To obtain other structures in the same P21 space group with two molecules per ASU (structures 2-7, 9, 15, 16) (see Supplementary Table 1-2), this test set from the apo dataset was carried over and used in refinement. After one round of rigid-body refinement using the apo structure (structure 1), iterative rounds of positional and individual B-factor refinements were then performed, with manual building and adjustments in Coot54 against 2mFo-DFc and composite omit maps contoured to 1.0 σ. Structure and geometric restraints for the CADA and CABA molecules and the vanadyl ion were generated using eLBOW56 using chemical SMILES strings (for CADA or CABA molecules) or the chemical component code VVO (for the vanadyl ion) as chemical inputs.

Since the F102Y apo structure 8 is in a different space group, C2, MR was used for phasing for this structure. Chain A of the refined apo-Rv0097 structure 1 was used for MR using Phaser-MR52 without modifications to the model. The final MR solution with LLG of 9877.1 and TFZ of 85.9 identified one protomer per ASU. This MR solution was directly used for refinements, first using rigid-body refinement, with 5% of total reflections randomly selected and set aside for the test set (Rfree). Following rigid-body refinement, iterative rounds of positional and individual B-factor refinements were then performed, with manual adjustments in Coot53 against 2mFo-DFc maps contoured to 1.0 σ. Waters were added and the structure was validated as described above.

The F102A apo structure 10 and the F102A-vanadyl-CADA structure 11 are in another new space group, P2221, therefore MR was used for phasing. Chain A of the refined apo-Rv0097 structure 1 was used for MR using Phaser-MR52 without modifications to the model. For the F102A apo structure 10, the final MR solution with LLG of 4580.2 and TFZ of 60.4 identified one protomer per ASU. This MR solution was directly used for refinements, first using rigid-body refinement, with 5% of total reflections randomly selected and set aside for the test set (Rfree). Following rigid-body refinement, iterative rounds of positional and individual B-factor refinements were then performed, with manual adjustments in Coot54 against 2mFo-DFc maps contoured to 1.0 σ. To obtain the F102A-vanadyl-CADA structure 11, refined model of the F102A apo structure 10 was used directly for refinement, starting with rigid-body refinement. The test reflection set for F102A apo structure 10 was carried over and used in the refinements for the F102A-vanadyl-CADA structure 11.

The rest of the CADA- and/or αKG-soaked structures 12, 13, 14 are in the P21 space group as most of the structures reported in this work, but their unit cell parameters are significantly different due to cell expansions caused by large conformational changes. Therefore, to obtain these structures, Chain A of the refined apo-Rv0097 structure 1 was used for MR using Phaser-MR52 without modifications to the model. We started with the 10 mM CADA soak dataset 13. The final MR solution with LLG of 16630.4 and TFZ 38.8 identified 4 protomers in the ASU. This MR solution was directly used for refinements, first using rigid-body refinement, with 5% of total reflections randomly selected and set aside for the test set (Rfree). This test set was carried over and used in refinements of the 5 mM CADA soak structure 12 and the 5 mM CADA and 5 mM αKG-soak structure 14. Following rigid-body refinement, iterative rounds of positional and individual B-factor refinements were then performed, with manual adjustments in Coot54 against 2mFo-DFc maps contoured to 1.0 σ. To obtain the 5 mM CADA soak structure 12 and the 5 mM CADA and 5 mM αKG-soak structure 14, refined model of the 10 mM CADA soak structure was used for refinement 13, starting from rigid-body refinement, followed by iterative rounds of positional and individual B-factor refinements and manual adjustments.

A summary of the data collection and refinement statistics for all reported structures can be found in Supplementary Table 1. All structural figures were generated using the PyMOL Molecular Graphics software package (Schrödinger, LLC). Crystallography package was compiled by SBGrid Consortium57.

Computational channel calculations
Channel prediction was performed by MOLEonline58 running MOLE version 2.5 using default parameters and with the atomic coordinates of structure 16, minus the side chain of R122.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors reported several high-resolution crystal structures of the isonitrile biosynthetic enzyme Rv0097 from\nMycobacterium tuberculosis, including both wild-type and variant forms in the presence and absence of substrate (CADA)\nand α-ketoglutarate (αKG). These structural insights help elucidate the enzyme’s architecture and suggest potential\nconformational changes that may contribute to isonitrile formation. However, the current manuscript lacks experimental\nevidence to substantiate the conclusions drawn from these structural snapshots. To this reviewer, it remains unclear whether\nthe observed conformation differences correspond to catalytically relevant intermediates or they are simply artifacts during\ncrystallization. In addition, several statements in the manuscript overlap with the authors’ previous work and other published\nliteratures. To merit publication in a high-impact journal such as Nature Communications, the authors should consider\ncarrying out experiments to provide insights into the proposed mechanism. Specific comments are listed below:\n1. For structures 3 (PDB ID: 9P9P, 1.48 Å), 5 (PDB ID: 9PAU, 1.7 Å), 9 (PDB ID: 9PBN, 1.46 Å), 10 (PDB ID: 9PBU, 1.73 Å),\n13 (PDB ID: 9PCL, 1.76 Å), and 15 (PDB ID: 9PCM, 1.45 Å), the reported R-values appear relatively high (Rwork/Rfree\n[structure 3] = 0.20/0.22; Rwork/Rfree [structure 5] = 0.21/0.24; Rwork/Rfree [structure 9] = 0.20/0.22; Rwork/Rfree [structure\n10] = 0.20/0.23; Rwork/Rfree [structure 13] = 0.20/0.24; Rwork/Rfree [structure 15] = 0.20/0.22) considering the resolution of\nthe datasets. The reviewer recommends that the authors re-examine the model coordinates and consider reprocessing the\ndiffraction data using an appropriate resolution cutoff to improve model quality and ensure coordinate reliability.\n2. The authors should clearly state whether the substrate 3-(carboxymethyl)amino decanoic acid (CADA) used in this study\nis a single isomer, e.g., C2-R, or a racemic mixture at the beta-position. The C3-chirality of CADA is not assigned in either\nScheme 1 or the Methods section. According to references 12 and 44 cited in the Methods, the starting material for CADA\nconsists of a mixture of R and S isomers rather than a single stereoisomer. If a racemic mixture was indeed used, this raises\nconcerns regarding the accuracy and interpretation of the reported kinetic parameters for CADA.\n3. To validate the importance of selected residues in substrate binding and specificity, the authors conducted enzyme\nactivity assays using CADA and determined kinetic parameters such as kcat and KM. In most cases, the variants exhibited\nreduced activity toward CADA. To gain insight into these variants, the authors should consider profiling the variants against\na series of substrates, e.g., those vary alkyl chain lengths. It is possible that the variants retain catalytic activity but alter the\nsubstrate preferences compared to the wild type. A similar approach has been demonstrated in a recent publication (RSC\nChem. Biol. 2025, 6, 583–589).\n4. The label of the y-axes in Figures 2H, 3I, and 4B is unclear and should be revised for better readability and interpretation.\nAdditionally, there appear to be errors in the variant names shown on the x-axes of these figures. The authors should\ncarefully check and correct the labeling to ensure consistency with the text and figure legends.\n5. The authors propose that, following the first half-reaction, the imine intermediate remains bound within the active site\nrather than undergoing dissociation and re-binding process. This is an important statement, but currently lacking\nexperimental support. Moreover, the kinetic data cited from references 11 and 14 raise questions about the proposed model.\nSpecifically, the reported KM value for the imine intermediate derived from CABA (KM = 369 ± 40 μM) is higher than that of\nthe parent substrate CABA (KM = 286 ± 93 μM). This suggests that the substrate binds slightly tighter than the intermediate,\nwhich does not support the proposed model. The authors should address this inconsistency, clarify how these kinetic values\nalign with their proposed mechanism, and provide additional experimental evidence to support their hypothesis.\n6. The authors discuss the concepts of online and offline 2OG binding, along with potential conformational changes, by\ncomparing their ternary structure (structure 15) with the Fe(IV)-oxo mimic vanadyl-bound structures. However, no side-by-\nside structural comparison figures are provided in the main text or SI. Without such comparisons, it is difficult for readers to\nevaluate the proposed differences in binding modes and associated conformational shifts. The authors are encouraged to\ninclude comparative figures to support and clarify their discussion.", "reviewer_2": "The authors report 16 structures of wild-type or selected variants of Rv0097, an intriguing isonitrile biosynthetic enzyme from\nMycobacterium tuberculosis. Significantly, the structures include those of the active Fe(II)-containing enzyme bound to\nalpha-ketoglutarate (aKG), to 3-carboxymethylaminodecanoic acid (CADA), and to both substrates. The results are\nconsistent with a reasonable two-step enzyme mechanism that includes an unstable imine intermediate, where each step\nrequires oxidative cleavage of aKG and involves conformational changes of several residues on two loops. The structures\nalso explain the preference of this enzyme for substrates possessing long alkyl groups in comparison to the preference for\nshort alkyl groups by ScoE, a related isonitrile-forming enzyme that was previously characterized structurally. The current\nstructural studies are nicely complemented by activity measurements of selected variants. The work is of high quality and\nshould be of great interest to many readers. The writing is logically presented and very clear, although numerous minor\nconcerns are present as indicated below:\nLine 47: Figure S1A shows the Trp nitrogen atom in red changing to blue in the product with the product carbon atom in red\nfrom an unclear carbon of Ru5P. An alternative coloration scheme could help a reader more clearly see the sources of the N\nand C atoms.\nLine 56: “These … synthesis” has inconsistent number agreement.\nLine 67: Figure S1B confusingly shows the N and C changing color during the reaction.\nLine 71: “ScoE/Rv0097 is” has inconsistent number agreement and would be better written as “ScoE and Rv0097 are”.\nLine 82: Figure S3 shows the C4 methyl group of butyrate transforming into a hydroxyl group rather than the glycyl moiety\nundergoing hydroxylation. Two of the structures in this figure are incorrect.\nLine 107: “apo” is lab jargon and should be defined at first use as the apoprotein.\nLine 108: It would be helpful to include the PDB ID for each structure in Table S1 despite Table S2 summarizing this\ninformation. State the method for calculating Rmerge in Table S1.\nLine 114: Delete “two” for clarity since there is only one His and one Arg of interest.\nLine 120: The legend of Figure S5 should merge two sentence fragments (“When modeled as a Mg ion. No mFo-DFc\ndifference…”) into a single sentence in line 3 and insert the word “be” into “to consistent” in line 4.\nLine 134: To contrast with the bulky F239 of ScoE It might help the reader see the increased space if G204 of Rv97 is\nincluded in Figure S6.\nLine 145: Perhaps specify the residues involved in the hydrophobic interactions with CADA in Figure 1E and clarify if they\nare conserved in ScoE.\nLine 148: Only DNA, not protein, is mutated so this line should be rewritten as “mutation of the codon for G204 to encode” or\n“substitution of G204”.\nLine 166: Can the authors provide an estimate for the occupancy of CABA?\nLine 168: Consider indicating that the data in panels A and B were obtained from triplicate measurements as mentioned in\nMethods? These are rate versus substrate concentration plots, not Michaelis-Menten kinetics plots (the plots were fit to\nMichaelis-Menton kinetics). The table sometimes includes an unrealistic number of significant digits (e.g. 8.07 +/- 1.57) and\nin some cases the number of significant digits in the value does not agree with the number of significant digits in the error\n(e.g. 1.72 +/- 0.4).\nLine 170: Perhaps define (as wild-type enzyme) this first use of “WT”.\nLine 176: The sentence is missing “of” after “roles”.\nLine 178: The sentence is misleading because CADA was not bound well to the F102A variant as implied for structure 11.\nPerhaps this sentence should indicate that the complexes with CADA also included vanadate. Perhaps Table S2 should\nindicate CADA was included for structure 11, even though it bound poorly.\nLine 222: The legend to Figure S9 should indicate these represent triplicate results and replace the phrase “Michaelis-\nMenten kinetics plot”.\nLine 227: Perhaps also state the substrate concentration so a reader will know if the enzyme kinetics studies used saturating\nconditions?\nLine 243: CADA soaking appears to not only crack or deform but also enlarge the crystals in Figure S10. Is this the case?\nLines 257 and 258: “half … undergoes”, not “half … undergo”.\nLine 278: Perhaps state the RMSD between the one open chain and the three closed states for the 10 mM CADA-soaked\nsample.\nLine 285: ScoE R157 is said to be equivalent to R122 of Rv0097 and several other comparable residues in these proteins\nare mentioned elsewhere. The authors should consider including a sequence alignment of the two proteins in the\nsupplementary material.\nLines 287 and 302: The terms offline and online when referring to aKG conformation should be defined\n(perhaps include a diagram?) and cite a reference.\nLine 344: The stated 1.45-A resolution for structure 16 differs from what is shown in Table S1 of 2.5-A.\nLines 418-420: This was the first time the manuscript mentions the previously published structural analysis of Rv0097. The\ntext should state the access code for that structure (8KHT), indicate it was for the complex of holoenzyme with substrate, and\ncompare the prior results to the holoenzyme + CADA structure reported here, perhaps where structure 2 is described.\nLine 477: insert “reactive” into “highly iron-oxygen intermediate”.\nLine 532: Table S5 should include the sources of the strains.\nLine 535: Define TB?\nLine 541: Define NTA?\nLine 556: WT should have been defined much earlier and can be used here without definition.\nLine 557: Consider changing “Reconstruction” to “Metallation”.\nLine 563-564: CADA and CABA were defined earlier and can be used without doing so again. Perhaps later in this\nparagraph the definitions could be provided for HRMS, NMR, HPLC?\nLine 595: Py-tetrazine was just defined in line 580; no need to duplicate.\nLine 586: INDA was already defined and can be used without redefinition.\nLines 612 and 614: Replace “media” with “medium”.\nLines 620 and 628: The g should be italicized.\nLine 635: EDTA was used earlier where the definition should be found so the definition is not needed here.\nLine 648: Perhaps state the initial crystallization screens?\nLine 671: Redefining aKG is not needed here. Also, portions of this and the following two paragraphs (e.g., “harvested, cryoprotected with paraffin oil, and flash frozen in liquid nitrogen”) are redundant; can the descriptions be simplified?\nLines 725, 735, 749, 760, and 777: The authors used 2|Fo|–|Fc| maps contoured to 1.0 σ for manual adjustment during\nmodel building. Why was this chosen instead of the more typical 2mFo-DFc maps?\nLines 726 and later: What were the contour levels of the 2mFo-DFc maps?\nLine 729 and later: Please state how the |Fo|–|Fc| maps at 3.0 σ were generated.\nLine 732: Was molecular replacement used for phasing structures 2-7, 9,15 and 16?\nLine 975: Consider showing the physiological protonation states for parts A and B. More importantly, the oxidation state of\niron in the hydroxyl intermediate is an issue. The hydroxylation of the glycyl group would result in an Fe(II) state of the\nenzyme. Is there evidence for coordination of the alkoxide to the metal ion?\nLine 982: Label D99 in Figure 1B.\nLine 985: Fe should be replaced by Mg.\nLine 986: It is mentioned on line 131, but it might be useful to restate here the occupancies of the two F102 rotamers.\nLine 987: Perhaps replace “only one” with “each single”?\nLine 995: Perhaps repeat the information from line 165 on the partial occupancy of CABA in panel H.\nLine 996: Please mention the map type and contour level for panels G, H and I.\nLine 998: The organization of the panels would be more logical by shifting panel B into the middle of the top row. The table\nin G has issues with the numbers of significant figures and in one case the agreement between the value and the error.\nThe y-axis label and values and the labels in the x-axis have issues in panel H. Also, the triplicate points should be shown, not\njust error bars.\nLine 1001: F102 should read F102W or W102.\nLines 1003-1009: Indicate the map type and contour level for panels 2B, C, D & E.\nLine 1011: Perhaps indicate the assay had used 1 mM substrate (far from saturating for F102A!) and was carried out for 10\nmin?\nLine 1014. The upper two number values on the Panel I y-axis and the sample labels (PA53A and PA53V) need attention.\nShowing the triplicate points would be appropriate.\nLine 1027: The centroid-to-centroid distance in Figure 3H is for the F102’, not F102, conformation in the open state.\nLine 1031: Panel B has errors in the y-axis title and labels as well as the names of the variants studied. Showing the\ntriplicate points would be a nice addition.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "The authors reported several high-resolution crystal structures of the isonitrile biosynthetic enzyme\nRv0097 from Mycobacterium tuberculosis, including both wild-type and variant forms in the presence\nand absence of substrate (CADA) and α-ketoglutarate (αKG). These structural insights help elucidate\nthe enzyme’s architecture and suggest potential conformational changes that may contribute to isonitrile\nformation.\nThank you very much for your comments.\nHowever, the current manuscript lacks experimental evidence to substantiate the conclusions drawn\nfrom these structural snapshots. To this reviewer, it remains unclear whether the observed conformation\ndifferences correspond to catalytically relevant intermediates or they are simply artifacts during\ncrystallization.\nThe reviewer is correct that crystal lattices can lead to artifacts. All too often the crystal lattice prevents\nmovements that are relevant to catalysis. However, Rv0097 is not a protein system that suffers from\nthis issue. We apologize if our writing of this manuscript did not make this clear. The reason that this\npaper has 16 structures is that Rv0097 crystallizes with lattices that are malleable to conformational\nchange, and when the lattice cannot accommodate the required conformational change, Rv0097\ncrystallized in different space groups. My lab has deposited over 200 entries to the Protein Data Bank,\nand I have never before worked on a protein system like Rv0097 that was so amenable to crystallization\nin so many different conformational states. We were incredibly fortunate in this regard. Additionally, our\nproposed mechanism based on these structural snapshots are further supported by biochemical studies\nand are in line with previous proposed mechanisms of related non-heme Fe(II)-αKG-dependent\ndioxygenase. We have edited the text to explain these points clearly.\nIn addition, several statements in the manuscript overlap with the authors’ previous work and other\npublished literatures.\nThank you for your comment. Our prior work on Rv0097 (Biochemistry 2023, 62, 3, 824–834) identified\nG204 residue as a putative residue gating substrate specificity. Later work by others (ACS Catal. 2024,\n14, 4975–4983 and RSC Chem. Biol. 2025, 6, 583–589) reported preliminary structural data identifying\nresidues in the substrate-binding pocket that govern substrate alkyl chain length specificity by\ndifferential side chain bulkiness. In this work, we revealed an additional layer of specificity control of\nRv0097 by a pair of substrate-binding site residues, F102/G204, that gate substrate binding and\nspecificity by maintaining a hydrophobic environment. We also report conformational changes of other\nregions of the enzyme that were not previously observed. This study showcases how multiple flexible\nregions achieve two half reactions in one catalytic cycle in a highly orchestrated fashion and highlights\nthe role of enzyme dynamics in controlling activities. We are building off of previous work. We have\nedited the text to make sure that it is clear as to what was known and what is newly discovered.\nTo merit publication in a high-impact journal such as Nature Communications, the authors should\nconsider carrying out experiments to provide insights into the proposed mechanism. Specific comments\nare listed below:\n1. For structures 3 (PDB ID: 9P9P, 1.48 Å), 5 (PDB ID: 9PAU, 1.7 Å), 9 (PDB ID: 9PBN, 1.46 Å), 10\n(PDB ID: 9PBU, 1.73 Å), 13 (PDB ID: 9PCL, 1.76 Å), and 15 (PDB ID: 9PCM, 1.45 Å), the reported R-\nvalues appear relatively high (Rwork/Rfree [structure 3] = 0.20/0.22; Rwork/Rfree [structure 5] =\n0.21/0.24; Rwork/Rfree [structure 9] = 0.20/0.22; Rwork/Rfree [structure 10] = 0.20/0.23; Rwork/Rfree\n[structure 13] = 0.20/0.24; Rwork/Rfree [structure 15] = 0.20/0.22) considering the resolution of the\ndatasets. The reviewer recommends that the authors re-examine the model coordinates and consider\nreprocessing the diffraction data using an appropriate resolution cutoff to improve model quality and\nensure coordinate reliability.\nrefinement against 2mF -DF omit maps.\no c\nAlthough truncating the data to lower resolution will lower R-factors, throwing away higher resolution\ndata cannot be justified in a case where the R-factors are appropriate for structures that are not over-\nrefined. The gaps between R and R are very small, indicating proper structure refinements with\nwork free\nno overfitting. Our current resolution cutoffs were determined based on holistic considerations of\nstatistics during data processing: R , CC , I/σ, and data completeness.\nsym 1/2\nAdditionally, R-factors lower than what we report here would not be expected for a small protein like\nRv0097 (with molecular weight of ~30 kDa) that has multiple regions that are flexible. Although we\nworked hard to model the flexible regions, using alternative conformations as appropriate, it is not\npossible to build a structural model that accounts for all of the protein's flexibility. Of course, all proteins\nhave at least one flexible loop and thus the agreement between Fobs and Fcalc is never perfect, but\nwhen a large percentage of a protein is flexible as is the case here, R-factors close to 20% is what one\nwould expect for structures that are not overrefined.\nAlthough we were satisfied with our R-factors, we did subject our structures to further refinement to\naddress this reviewers' concern. We performed an additional final round of Translation-Libration-Screw\n(TLS) refinement for each of the above-mentioned structures. The R-factors are lowered for some\nstructures (structures 3, 9, and 10) whereas they stayed roughly the same for the others. The table\nbelow summarizes these changes in R-factors, and the new R-factors are now added to Table S1. We\nexamined the maps individually before and after TLS refinements and found no noticeable differences.\nPlease also see screenshots below.\nR-factors before and after a final round of TLS refinement\nStructure Resolution (Å) Before TLS After TLS refinement\nrefinement (current)\nR /R\nwork free\nR /R\nwork free\n3 1.48 0.199/0.219 0.179/0.195\n5 1.70 0.211/0.236 0.205/0.232\n9 1.46 0.195/0.218 0.175/0.192\n10 1.73 0.195/0.228 0.177/0.210\n13 1.76 0.198/0.237 0.194/0.230\n15 1.45 0.195/0.218 0.193/0.215\nThe following images are screenshots of 2mF -DF maps (1.5 σ, blue mesh) and mF -DF maps (+3.0\no c o c\nσ, green mesh, -3.0 σ, red mesh) with atomic models of structures 3, 9, and 10 fit in before and after\none final round of TLS refinement:\n2\n2. The authors should clearly state whether the substrate 3-(carboxymethyl)amino decanoic acid\n(CADA) used in this study is a single isomer, e.g., C2-R, or a racemic mixture at the beta-position. The\nC3-chirality of CADA is not assigned in either Scheme 1 or the Methods section. According to references\n12 and 44 cited in the Methods, the starting material for CADA consists of a mixture of R and S isomers\nrather than a single stereoisomer. If a racemic mixture was indeed used, this raises concerns regarding\nthe accuracy and interpretation of the reported kinetic parameters for CADA.\nIn this study, we utilized a racemic mixture of CADA (at the β-position) for the biochemical\ncharacterization of Rv0097, as detailed in our previous publication in Biochemistry (DOI:\n10.1021/acs.biochem.2c00611). The current version of the manuscript has been revised to explicitly\nclarify this point.\nRegarding the use of a racemic mixture of CADA, we did consider synthesizing the enantiomerically\npure R-isomer of CADA. However, this approach would require a significant investment of time\n(estimated at over 2–3 months) and incur substantial material costs (exceeding $3,000 with an\nestimated lead time of 30 days). While we recognize that using enantiomerically pure CADA would be\nideal, we are confident in the validity of our kinetic parameters. All enzyme variants were assessed\n3\nusing the same R/S mixture of CADA, thus allowing for reliable comparative analysis across the different\nenzyme variants.\n3. To validate the importance of selected residues in substrate binding and specificity, the authors\nconducted enzyme activity assays using CADA and determined kinetic parameters such as kcat and\nKM. In most cases, the variants exhibited reduced activity toward CADA. To gain insight into these\nvariants, the authors should consider profiling the variants against a series of substrates, e.g., those\nvary alkyl chain lengths. It is possible that the variants retain catalytic activity but alter the substrate\npreferences compared to the wild type. A similar approach has been demonstrated in a recent\npublication (RSC Chem. Biol. 2025, 6, 583–589).\nWe agree with the reviewer that profiling the variants against different substrates is important to probe\nresidues potentially affecting chain length recognition, which has been extensively studied by us, by\ncomparing CADA vs. CABA (this manuscript and Biochemistry 2023, 62, 824-834), and other\nresearchers (ACS Catal. 2024, 14, 4975-4983; RSC Chem. Biol. 2025, 6, 583–589). However, we feel\nthat our kinetic analysis toward CADA is sufficient to probe the role of the F102/G204 pair in governing\nsubstrate binding and specificity, the role of P153 in gating CADA binding, and the role of H264/R122\nin gating binding of co-substrates.\n4. The label of the y-axes in Figures 2H, 3I, and 4B is unclear and should be revised for better readability\nand interpretation. Additionally, there appear to be errors in the variant names shown on the x-axes of\nthese figures. The authors should carefully check and correct the labeling to ensure consistency with\nthe text and figure legends.\nThank you for pointing this issue out. The problem arose in the PDF conversion that was performed on\nthe journal website. I am not sure how to fix this problem since the issue does not arise when the PDF\nis viewed on a home computer. We remade these figure panels with the hope of improved compatibility\nand will check the conversion before submission.\n5. The authors propose that, following the first half-reaction, the imine intermediate remains bound\nwithin the active site rather than undergoing dissociation and re-binding process. This is an important\nstatement, but currently lacking experimental support. Moreover, the kinetic data cited from references\n11 and 14 raise questions about the proposed model. Specifically, the reported KM value for the imine\nintermediate derived from CABA (KM = 369 ± 40 μM) is higher than that of the parent substrate CABA\n(KM = 286 ± 93 μM). This suggests that the substrate binds slightly tighter than the intermediate,\nwhich does not support the proposed model. The authors should address this inconsistency, clarify how these\nkinetic values align with their proposed mechanism, and provide additional experimental evidence to\nsupport their hypothesis.\nTo investigate whether the imine-CADA intermediate remains sequestered within the enzyme active\nsite, we have performed an additional series of biochemical assays employing a previously established\nmethod for the indirect detection of the imine via chemical derivatization of glyoxylate under acidic\nconditions (DOI: 10.1021/jacs.1c12891). Reactions were quenched after short incubation periods (1\nminute), followed by chemical derivatization to assess glyoxylate formation for imine-CADA intermediate\nquantification. To distinguish between free and enzyme-bound imine-CADA, we compared the relative\namounts in the reaction supernatant alone (enzyme is removed via filtration) to that of the complete\nreaction mixture containing the enzyme (see new SI Figure, Fig. S20). The results from this new\nexperiment show substantially more imine-CADA in the sample with the enzyme present, consistent\nwith imine-CADA remaining bound in the active site during catalysis, and consistent with imine-CADA\nhaving a relatively high affinity for the enzyme. The somewhat higher K for the imine-CADA compared\nM\nto CADA does not conflict with our proposal that the imine-CADA remains bound to the enzyme during\ncatalysis. A somewhat higher K merely indicates that imine-CADA is a slightly poorer substrate than\nM\nCADA if added exogenously. The new data inform on binding affinity of imine-CADA rather than on its\nK and are a nice addition to the paper. We thank the reviewer for encouraging us to perform additional\nM\nexperiments.\nnew SI figure (Fig. S20).\n6. The authors discuss the concepts of online and offline 2OG binding, along with potential\nconformational changes, by comparing their ternary structure (structure 15) with the Fe(IV)-oxo mimic\nvanadyl-bound structures. However, no side-by-side structural comparison figures are provided in the\nmain text or SI. Without such comparisons, it is difficult for readers to evaluate the proposed differences\nin binding modes and associated conformational shifts. The authors are encouraged to include\ncomparative figures to support and clarify their discussion.\nThank you for your suggestion. We have now added a new SI figure (Fig. S16) that provides these side-\nby-side comparisons.\nReviewer #2 (Remarks to the Author):\nThe authors report 16 structures of wild-type or selected variants of Rv0097, an intriguing isonitrile\nbiosynthetic enzyme from Mycobacterium tuberculosis. Significantly, the structures include those of the\nactive Fe(II)-containing enzyme bound to alpha-ketoglutarate (aKG), to 3-carboxymethylaminodecanoic\nacid (CADA), and to both substrates. The results are consistent with a reasonable two-step enzyme\nmechanism that includes an unstable imine intermediate, where each step requires oxidative cleavage\nof aKG and involves conformational changes of several residues on two loops. The structures also\nexplain the preference of this enzyme for substrates possessing long alkyl groups in comparison to the\npreference for short alkyl groups by ScoE, a related isonitrile-forming enzyme that was previously\ncharacterized structurally. The current structural studies are nicely complemented by activity\nmeasurements of selected variants. The work is of high quality and should be of great interest to many\nreaders. The writing is logically presented and very clear, although numerous minor concerns are\npresent as indicated below:\nThank you very much for your very detailed comments and suggestions that helped us greatly to\nimprove the quality of our manuscript.\nLine 47: Figure S1A shows the Trp nitrogen atom in red changing to blue in the product with the product\ncarbon atom in red from an unclear carbon of Ru5P. An alternative coloration scheme could help a\nreader more clearly see the sources of the N and C atoms.\nCorrected.\nLine 56: “These … synthesis” has inconsistent number agreement.\nCorrected.\nLine 67: Figure S1B confusingly shows the N and C changing color during the reaction.\nCorrected.\nLine 71: “ScoE/Rv0097 is” has inconsistent number agreement and would be better written as “ScoE\nand Rv0097 are”.\nChanged to \"ScoE and its homologs are...\"\nLine 82: Figure S3 shows the C4 methyl group of butyrate transforming into a hydroxyl group rather\nthan the glycyl moiety undergoing hydroxylation. Two of the structures in this figure are incorrect.\nCorrected. Thank you.\nLine 107: “apo” is lab jargon and should be defined at first use as the apoprotein.\n5\n“Apo” is now first defined as apoprotein.\nLine 108: It would be helpful to include the PDB ID for each structure in Table S1 despite Table S2\nsummarizing this information. State the method for calculating Rmerge in Table S1.\nPDB IDs for each structure are now added to Table S1. Per standard formatting guidelines for Nature\njournals, equations defining various R-values are standard and hence are no longer defined in the\nfootnotes (https://www.nature.com/nature/for-authors/formatting-guide).\nLine 114: Delete “two” for clarity since there is only one His and one Arg of interest.\nCorrected.\nLine 120: The legend of Figure S5 should merge two sentence fragments (“When modeled as a Mg ion.\nNo mFo-DFc difference…”) into a single sentence in line 3 and insert the word “be” into “to consistent”\nin line 4.\nCorrected.\nLine 134: To contrast with the bulky F239 of ScoE It might help the reader see the increased space if\nG204 of Rv97 is included in Figure S6.\nFig. S6 is now updated to include G204 (α carbon shown as a sphere) of Rv0097.\nLine 145: Perhaps specify the residues involved in the hydrophobic interactions with CADA in Figure\n1E and clarify if they are conserved in ScoE.\nWe added a list of residues involved in hydrophobic interactions with CADA. We also added a sequence\nalignments SI figure (Fig. S7) highlighting these residues across Rv0097 homologs: ScoE and MmaE.\nWe additionally added an SI figure (Fig. S9) showing that two loops in the substrate-binding pocket are\npositioned differently in ScoE and Rv0097 to accommodate the different substrate alkyl chain lengths.\nLine 148: Only DNA, not protein, is mutated so this line should be rewritten as “mutation of the codon\nfor G204 to encode” or “substitution of G204”.\nCorrected.\nLine 166: Can the authors provide an estimate for the occupancy of CABA?\nYes. Refined occupancy of CABA is 0.65 in this structure. We have added this information to the text.\nLine 168: Consider indicating that the data in panels A and B were obtained from triplicate\nmeasurements as mentioned in Methods? These are rate versus substrate concentration plots, not\nMichaelis-Menten kinetics plots (the plots were fit to Michaelis-Menton kinetics). The table sometimes\nincludes an unrealistic number of significant digits (e.g. 8.07 +/- 1.57) and in some cases the number\nof significant digits in the value does not agree with the number of significant digits in the error\n(e.g. 1.72 +/- 0.4).\nWe have added in the figure legend of Fig. S10 (old Fig. S8) that data in panels A and B were obtained\nfrom triplicate measurements. We have edited the legends to be velocity vs. substrate concentration\nplots. The tables now include values with correct numbers of significant digits, and that the number of\nsignificant digits of error are consistent with the number of significant digits of the values. We also made\nsure that errors of k /K are calculated following the rules for propagation of uncertainty.\ncat M\nLine 170: Perhaps define (as wild-type enzyme) this first use of “WT”.\nWT is now first defined as wild-type enzyme.\nLine 176: The sentence is missing “of” after “roles”.\nLine 178: The sentence is misleading because CADA was not bound well to the F102A variant as\nimplied for structure 11. Perhaps this sentence should indicate that the complexes with CADA also\nincluded vanadate. Perhaps Table S2 should indicate CADA was included for structure 11, even though\nit bound poorly.\nWe edited the text and Table S2 as suggested.\nLine 222: The legend to Figure S9 should indicate these represent triplicate results and replace the\nphrase “Michaelis-Menten kinetics plot”.\nWe have added in the figure legend of Fig. S11 (old Fig. S9) that data in panels A and B were obtained\nfrom triplicate measurements. We have edited the legends to be velocity vs. substrate concentration\nplots.\nLine 227: Perhaps also state the substrate concentration so a reader will know if the enzyme kinetics\nstudies used saturating conditions?\nSubstrate concentration for assay (1 mM) is added. This is not saturating concentration – we use the\nterm “saturating” here per definition of k as we discuss and compare k values of different enzyme\ncat cat\nvariants.\nLine 243: CADA soaking appears to not only crack or deform but also enlarge the crystals in Figure\nS10. Is this the case?\nCrystals are not enlarged. Sorry, the photos are misleading in this regard. Photos of crystals before and\nafter the soak are zoomed-in differently. Deformed crystals are more zoomed in to show the cracks\nmore clearly. We have added this information in the figure legend.\nLines 257 and 258: “half … undergoes”, not “half … undergo”.\nCorrected.\nLine 278: Perhaps state the RMSD between the one open chain and the three closed states for the 10\nmM CADA-soaked sample.\nWe have added RMSD values in the paragraph describing the overlayed open and closed structures.\nLine 285: ScoE R157 is said to be equivalent to R122 of Rv0097 and several other comparable residues\nin these proteins are mentioned elsewhere. The authors should consider including a sequence\nalignment of the two proteins in the supplementary material.\nWe added a new SI figure (Fig. S7) providing sequence alignment of Rv0097, ScoE, and MmaE,\nhighlighting these residues.\nLines 287 and 302: The terms offline and online when referring to aKG conformation should be defined\n(perhaps include a diagram?) and cite a reference.\nDefinitions of offline have been added to the text. We included a new SI figure (Fig. S16) to show online\nand offline KG/Fe(IV)-oxo conformations.\nLine 344: The stated 1.45-A resolution for structure 16 dimers from what is shown in Table S1 of 2.5-A.\nThe resolution of structure 16 is 1.36 Å (not 1.45 Å). We have fixed this value in the text. It was correct\nin Table S1. Thank you for noticing this typo. The resolution of the anomalous data for this structure\nwas 2.5 Å.\nLines 418-420: This was the first time the manuscript mentions the previously published structural\nanalysis of Rv0097. The text should state the access code for that structure (8KHT), indicate it was for\n7\nthe complex of holoenzyme with substrate, and compare the prior results to the holoenzyme + CADA\nstructure reported here, perhaps where structure 2 is described.\nWe now reference this structure in the introduction. Additionally, the PDB accession code 8KHT is also\nadded to lines 418-420. A structural similarity comparison is added to the location in the text where\nstructure 2 is first mentioned.\nLine 477: insert “reactive” into “highly iron-oxygen intermediate”.\nCorrected.\nLine 532: Table S5 should include the sources of the strains.\nSources of strains have been added.\nLine 535: Define TB?\nTB defined.\nLine 541: Define NTA?\nNTA defined.\nLine 556: WT should have been defined much earlier and can be used here without definition.\nWT is now defined earlier and the definition here is removed.\nLine 557: Consider changing “Reconstruction” to “Metallation”.\nWe changed “Reconstruction” to “Reconstitution” to be consistent with terms we used in our previous\nworks.\nLine 563-564: CADA and CABA were defined earlier and can be used without doing so again. Perhaps\nlater in this paragraph the definitions could be provided for HRMS, NMR, HPLC?\nDefinitions of CADA and CABA here are removed. HRMS, NMR, HPLC are defined.\nLine 595: Py-tetrazine was just defined in line 580; no need to duplicate.\nDuplicate definition is removed.\nLine 586: INDA was already defined and can be used without redefinition.\nWe removed “isonitrile” and now the sentence reads “The production of INDA…”\nLines 612 and 614: Replace “media” with “medium”.\nCorrected.\nLines 620 and 628: The g should be italicized.\nCorrected.\nLine 635: EDTA was used earlier where the definition should be found so the definition is not needed\nhere.\nEDTA is now first defined in the first paragraph of Results section and the following duplicate definitions\nare removed.\nLine 648: Perhaps state the initial crystallization screens?\nInitial crystallization screen added.\n(e.g., “harvested, cryoprotected with paraffin oil, and flash frozen in liquid nitrogen”) are redundant; can\nthe descriptions be simplified?\nαKG is defined here to indicate the nature of the salt and to give source of commercially purchased\nαKG used. We have trimmed the redundancy throughout the methods.\nLines 725, 735, 749, 760, and 777: The authors used 2|Fo|–|Fc| maps contoured to 1.0 σ for manual\nadjustment during model building. Why was this chosen instead of the more typical 2mFo-DFc maps?\nThe maps we used were indeed σ -weighted 2mF -DF and mF -DF maps for model building in Coot\nA o c o c\n(outputs of phenix.refine). We now specifically indicate that the maps were weighted.\nLines 726 and later: What were the contour levels of the 2mFo-DFc maps?\nWe have double checked that contour levels are provided.\nLine 729 and later: Please state how the |Fo|–|Fc| maps at 3.0 σ were generated.\nAll 2mF -DF and mF -DF maps are generated by phenix.refine. This information is now added.\no c o c\nLine 732: Was molecular replacement used for phasing structures 2-7, 9,15 and 16?\nNo. When the space group was the same, molecular replacement was not needed. We started with\nrigid-body refinement using the apo structure 1. This information is already included in the text.\nLine 975: Consider showing the physiological protonation states for parts A and B. More importantly,\nthe oxidation state of iron in the hydroxyl intermediate is an issue. The hydroxylation of the glycyl group\nwould result in an Fe(II) state of the enzyme. Is there evidence for coordination of the alkoxide to the\nmetal ion?\nWe updated Scheme 1A and 1B. with relevant protonation states of molecules. We do not have\nevidence for alkoxide coordination to the iron. We replaced the alkoxide species with the hydroxylated\nintermediate and corrected the oxidation state of iron to Fe(II).\nLine 982: Label D99 in Figure 1B.\nD99 labeled.\nLine 985: Fe should be replaced by Mg.\nCorrected.\nLine 986: It is mentioned on line 131, but it might be useful to restate here the occupancies the two\nF102 rotamers.\nF102 rotamer occupancies added.\nLine 987: Perhaps replace “only one” with “each single”?\nWe changed to \"a single\".\nLine 995: Perhaps repeat the information from line 165 on the partial occupancy of CABA in panel H.\nAdded.\nLine 996: Please mention the map type and contour level for panels G, H and I.\nMap information included.\nLine 998: The organization of the panels would be more logical by shifting panel B into the middle of\nthe top row. The table in G has issues with the numbers of significant figures and in one case the\n9\nagreement between the value and the error. The y-axis label and values and the labels in the x-axis\nhave issues in panel H. Also, the triplicate points should be shown, not just error bars.\nWe have reorganized panels in Fig. 2 so that each row shows one variant. Significant figures in Fig.\n2G have been fixed. Some of the issues with Fig. 2H have to do with the journal's PDF converter and\nare out of our control, but we did add individual data points in addition to the error bars.\nLine 1001: F102 should read F102W or W102.\nCorrected.\nLines 1003-1009: Indicate the map type and contour level for panels 2B, C, D & E.\nWe were trying to avoid redundancy but now repeat the map information in each panel description.\nLine 1011: Perhaps indicate the assay had used 1 mM substrate (far from saturating for F102A!) and\nwas carried out for 10 min?\nWe have added this information.\nLine 1014. The upper two number values on the Panel I y-axis and the sample labels (PA53A and\nPA53V) need attention. Showing the triplicate points would be appropriate.\nMost of the issues with Figure 3I were caused by the Journal's PDF converter, but we have added\nindividual data points in addition to the error bars. We also remade the panel in the hope of improving\ncompatibility with the PDF converter.\nLine 1027: The centroid-to-centroid distance in Figure 3H is for the F102’, not F102, conformation in the\nopen state.\nCorrected.\nLine 1031: Panel B has errors in the y-axis title and labels as well as the names of the variants studied.\nShowing the triplicate points would be a nice addition.\nMost of the issues with Figure 4B were caused by the Journal's PDF converter, but we have added\nindividual data points in addition to the error bars. We also remade the panel in the hope of improving\ncompatibility with the PDF converter.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nThank you for assisting in this review.\nReviewer #1 (Remarks to the Author):\nThis reviewer appreciates the authors’ detailed clarification and recognizes their extensive expertise in\nstructural biology and crystallization. The explanation addresses the concern regarding potential lattice\nartifacts and highlights the exceptional crystallographic behavior of Rv0097. However, the biochemical\nevidence obtained using the racemic substrate, racemic-CADA, remains unsolved and raises several\nconcerns that could impact the conclusion drown in this manuscript.\nThank you.\nEnantiomerically pure CADA has been synthesized on a gram scale in several reported literature\nincluding PNAS, 2022, 119, e2110293119; ACS Catal. 2024, 14, 4975−4983; J. Am. Chem. Soc. 2025\nDOI: 10.1021/jacs.5c13262. Therefore, the use of a racemic compound should not be considered as a\ntechnical limitation. Unless the authors clearly establish that the S-isomer does not influence the kinetic\nmeasurements or the interpretation of intermediate retention within the active site, the current data they\npresent seem unclear to support the conclusion. Specifically, it should be clarified whether the S-isomer\nis a competent substrate for Rv0097, and if so, whether it proceeds to the isonitrile product or instead\nlead to hydroxylated or imine intermediate formation. Furthermore, given that authors emphasize the\nrole of Rv0097 variants in governing substrate binding and specificity, it is important to address whether\nthese variants can affect the binding affinity or reactivity of the S-isomer.\nPrevious work (ACS Catal. 2024, 14, 4975–4983, ref. 26) showed that the S-isomer of CADA has\nnegligible activity for isonitrile formation by Rv0097 compared to the R-isomer. The authors observed\nover 20-fold decrease in enzymatic activity when S-isomer is used as the substrate. We have now\nprovided additional statements in the Results section that explicitly reference this work. In addition, we\nclearly observe R-CADA binding in the active site in all our CADA-bound WT and variant crystal\nstructures despite the use of the racemic mixture, suggesting weak competition from S-CADA. We\nmention this fact explicitly in the Results:\nLines 138—141: We note here that we used racemic-CADA for co-crystallization due to substantial\ncosts and time investments it requires to synthesize the enantiopure (R)-CADA26. We find only the (R)\nisomer bound in the structure, indicating the preference of the enzyme for (R)-CADA over (S)-CADA,\nas has been previously reported26.\nWe also now acknowledge in the Results that the use of a racemic mixture of CADA is a caveat that\nshould be keep in mind:\nLines 176—180: As with the structural studies, we used a racemic mixture of CADA in all our assays.\nSince we don't see evidence of (S)-CADA binding in our structures, we believe that the impact of the\nracemic CADA mixture on enzyme activity is minor, consistent a with previous report26. However, the\nuse of a racemic mixture of CADA and enantiomeric pure CABA is a caveat that should be kept in mind.\nMoreover, the authors should acknowledge relevant contributions from other researchers and\nincorporate the references in the main text, rather than citing them only in the response letter. To merit\npublication in a high-impact journal, the authors are encouraged to clearly address the issues outlined\nabove.\nThank you. All references we cited in our previous response letter were cited in the main text except for\none, which was an oversight. We now include it as new ref. 42:\nto the discovery of novel isonitrilases and decodes their substrate selectivity. RSC Chem. Biol.\n6, 583–589 (2025). https://doi.org/10.1039/D4CB00304G\nReviewer #2 (Remarks to the Author):\nOne minor point to fix is in the legend to Figure S5; the word \"density\" is crossed out but should be deleted.\nWe fixed this typo. Thank you for catching it.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nThank you for assisting in this review.\n2"}]

Decision letter