Trifunctional flavoenzyme-catalyzed asymmetric 4-alkyl-butenolide assembly in avenolide biosynthesis

Domain: neuroscience
Published: 2026-02-07
Editorial outcome: minor_revision

Abstract
Butenolides are important features of many bioactive compounds. Current petrochemical-based methods for asymmetric butenolide synthesis are unsustainable. Biocatalysis could enable cheaper, greener, and more sustainable alternatives. However, known butenolide biosynthetic pathways are complex and challenging to harness. Here we show that avenolide, a 4-alkylbutenolide that regulates avermectin production in Streptomyces avermitilis, is assembled from a fatty acyl thioester by a multifunctional flavoenzyme and an iterative cytochrome P450. The flavoenzyme catalyzes homochiral 4-alkylbutenolide formation via successive desaturation, hydroxylation, and lactonization reactions, using dioxygen as the sole stoichiometric reagent. This lays the foundation for development of sustainable biocatalytic asymmetric butenolide synthesis.

Introduction
Butenolides, featuring an α,β-unsaturated five-membered lactone, are structural features of numerous biologically active natural products, and important pharmacophores in several synthetic drugs (Fig. 1). The electrophilicity of the α,β-unsaturated ester (as a Michael acceptor) and the nucleophilicity of the dienolate resulting from γ-deprotonation make butenolides versatile building blocks for the construction of diverse molecular architectures1,2. Butenolides have thus attracted widespread attention from the organic synthesis community.

Two general synthetic strategies for butenolide synthesis have been developed (Supplementary Fig. 1). One assembles the core skeleton by intramolecular (e.g. ring closure metathesis, hetero-Pauson-Khand reaction) or intermolecular (e.g. acylation-Wittig olefination, [3+2] cycloaddition) cyclisation3–6. The other relies on functionalization of elaborated furanone precursors through Mukaiyama-Michael addition or palladium-catalyzed alkylation7–9. Both approaches involve multiple synthetic steps and reagents, making them laborious, expensive, and unattractive from a sustainability perspective.

Although an array of structurally diverse butenolide-containing natural products has been discovered (Fig. 1), biosynthetic mechanisms for butenolide formation remain relatively poorly characterized. The main pathways for butenolide formation characterized to date involve condensation of fatty acid/polyketide-derived β-keto thioesters with various 3-carbon glycolytic intermediates, followed by diverse tailoring reactions (Supplementary Fig. 2). They typically involve several enzymes with diverse co-factor requirements, rendering the development of efficient biocatalytic asymmetric approaches to 4-substituted butenolide synthesis challenging.

Avenolide contains a 4-alkylbutenolide and is a diffusible hormone that induces the avermectin production in Streptomyces avermitilis (Fig. 2a)10,11. Avermectins are a group of 16-membered macrolides with potent anthelmintic and insecticidal activity12. Both naturally occurring avermectins and semisynthetic derivatives (e.g. ivermectin) are widely used in veterinary/human medicine and agriculture to treat parasitic infections and deter insect pests13,14. Their biological activity stems from binding to non-mammalian glutamate-gated chloride ion channels, causing paralysis and death15.

Diffusible hormones containing or derived from butenolides are widespread in Streptomyces and related actinomycete genera. They bind to specific transcriptional repressors with nanomolar affinity, triggering the production of diverse specialized metabolites16,17. All classes of such hormones biosynthetically-characterized to date, including γ-butyrolactones, 2-alkyl-4-hydroxymethyl-furan-3-carboxylic acids, and 2-akyl-4-hydroxy-3-methyl-butenolides, are known to derive from analogous phosphorylated butenolide intermediates formed via the phosphorylated butenolide synthase (PBS)-catalyzed condensation of fatty acid-derived β-keto thioesters with dihydroxyacetone phosphate (Supplementary Fig. 2a)18–20.

In contrast, the biosynthesis of avenolide does not involve a PBS. Instead, enzymes with sequence similarity to acyl-CoA dehydrogenases (ACADs)/acyl-CoA oxidases (AOXs) and cytochromes P450 (CYPs), encoded by savA and savB (previously named aco and cyp17), respectively, have been proposed to assemble avenolide (Fig. 2a). Deletion of savA abolishes the production of avenolide, resulting in a significant decrease in avermectin production compared to the wild type strain10. However, the precise roles played by SavA and SavB in avenolide biosynthesis remain elusive.

Here we report elucidation of the roles played by SavA and SavB in avenolide biosynthesis. SavA catalyzes a unique desaturation-hydroxylation-lactonization cascade, converting a medium-chain fatty acyl thioester into the corresponding homochiral 4-alkylbutenolide. In vitro enzymatic assays using chemically synthesized substrates and product standards, 18O labelling, and SavA active site mutants suggest a mechanism for butenolide formation involving FAD-mediated α,β-desaturation, FADH2-mediated γ-hydroxylation, and spontaneous lactonization. Intriguingly, dioxygen is the only stoichiometric reagent; the flavin cofactor plays a purely catalytic role. Avenolide assembly is completed by SavB, which catalyzes three sequential regio/stereospecific hydroxylations of the alkyl chain.

Results
Heterologous expression of savAB in Streptomyces albidoflavus
Bacterial hormones usually work at nanomolar concentrations, making it difficult to obtain sufficient quantity from native producer strains for structure elucidation. Previously, 1.2 mg of avenolide was isolated and characterized from a 2000 L culture of S. avermitilis, the industrial avermectin producer10. This low titer represents a formidable challenge to biosynthetic studies in the native producer. It has been proposed that savA and savB, encoding homologs of an ACAD/AOX and a CYP, respectively, are responsible for the assembly of avenolide10. We therefore elected to examine the expression of savAB in several heterologous Streptomyces hosts, including Streptomyces coelicolor M1152, Streptomyces lividans TK24 and Streptomyces albidoflavus J1074. Homologs of savA and savB (XNR_2339 and XNR_2340), are present in the genome of S. albidoflavus J1074, which produces several butenolides structurally related to avenolide21. To avoid the possibility of these genes interfering with the expression of savAB, we deleted XNR_2339 and XNR_2340 in S. albidoflavus J1074 to generate S. albidoflavus M1. A pSET152 derivative containing the savAB genes under the control of the strong constitutive ermE* promoter was constructed and integrated into the chromosome of S. coelicolor M1152, S. lividans TK24, and S. albidoflavus M1 (Fig. 3a).

Although expression of savAB in most of the strains tested failed to result in detectable avenolide production, a prominent new metabolite was observed in UV-HPLC analyses of the culture extracts of S. albidoflavus M1 containing these genes (Fig. 3b). This metabolite was not present in culture extracts of S. albidoflavus M1 containing the empty vector and gave rise to an ion with m/z=241.1430, corresponding to [M+H]+ of avenolide (m/z calculated for C13H21O4+: 241.1434), in LC-MS analyses. This compound was isolated by preparative HPLC from ethyl acetate extracts of the culture broth of S. albidoflavus M1/pSET152::savAB grown in AlaMM medium for seven days. 8–10 mg of the compound were typically obtained from a 1 L culture. NMR spectroscopic data for the purified compound were identical to those previously reported for avenolide isolated from S. avermitilis (Supplementary Fig. 3)10. This confirmed that savA and savB together direct avenolide biosynthesis. Furthermore, the biological activity of avenolide purified from S. albidoflavus M1/pSET152::savAB was demonstrated in vitro and in vivo. An electrophoretic mobility shift assay (EMSA) demonstrated the ability of the compound to release AvaR1 from its operator and addition of the compound to a savA mutant of S. avermitilis restored avermectin production to wild-type levels (Supplementary Fig. 4).

The structural features of avenolide prompted us to hypothesize that the carbon skeleton of the molecule derives from a 10-methyl-dodecanoyl thioester intermediate in fatty acid biosynthesis via a dehydrogenation-hydroxylation-cyclisation cascade and three successive hydroxylation reactions. The desaturation reaction required to form the cis double bond in the butenolide was proposed to be catalyzed by SavA, which is homologous to ACADs/AOXs. Such enzymes introduce α,β-trans double bonds into acyl-CoA thioesters during each cycle of fatty acid β-oxidation in peroxisomes or mitochondria (Fig. 2b)22,23. FAD is an essential co-factor for these enzymes. SavB, which is homologous to CYPs, was proposed to catalyze three successive hydroxylation reactions at C-9 and C-10 of the 10-methyl-dodecanoyl thioester. We considered two alternative scenarios (Fig. 2a) for butenolide formation: (i) in addition to C-9 and C-10, SavB catalyzes hydroxylation at C-4, enabling subsequent lactonization, or (ii) SavA is multifunctional and catalyzes C-4 hydroxylation and lactonization, in addition to cis double bond formation.

CYPs typically use O2 as a co-substrate, utilizing it to produce a reactive ferryl-oxo complex that inserts an oxygen atom into a C-H bond of the substrate (Fig. 2c)24. To examine the involvement of O2 in avenolide biosynthesis, the S. albidoflavus M1/pSET152::savAB was grown under an 18O2 atmosphere. High-resolution MS analysis of the resulting avenolide showed that up to three 18O atoms were incorporated into its structure (Fig. 3c). No labeling of avenolide was observed in a control experiment utilizing air in place of 18O2. These data indicated that three of the four oxygen atoms in avenolide derive from O2. Thus, it seemed likely that SavB not only catalyzes hydroxylation of C-9 and C-10 in the 10-methyl-dodecanoyl thioester, but may also contribute to butenolide formation. However, further experiments were needed to fully define the roles played by SavA and SavB in avenolide biosynthesis.

SavA assembles the butenolide from a medium chain thioester
Sequence analyses suggested that SavA contains an N-terminal domain with homology to mitochondrial ACAD and a C-terminal domain showing similarity to peroxisomal AOX. Both enzymes catalyze FAD-dependent α,β-dehydrogenation of acyl-CoAs, yielding the corresponding trans-enoyl-CoAs (Fig. 2b)23.

To elucidate the function of SavA, we cloned and overexpressed the corresponding gene in Escherichia coli BL21 Star (DE3). Recombinant SavA was produced as an N-terminal His6-tagged fusion protein and purified to homogeneity using nickel affinity chromatography (Supplementary Fig. 5). The identity of the purified protein was confirmed by UHPLC-ESI-Q-TOF-MS. The protein exhibited an intense yellow color in solution, indicative of a bound flavin. Its UV-visible absorbance spectrum also displayed the typical features of an oxidized flavin, with major absorbance maxima at 360 and 446 nm. HPLC comparison of an extract from denatured SavA with an authentic standard of FAD further confirmed that the bound cofactor is FAD.

To identify the substrate of SavA, we chemically synthesized 10-methyl-dodecanoic acid 5 in three steps from 8-bromo-octanoic acid. Coupling of 5 with protected pantetheine 6 and subsequent hydrolysis, yielded the corresponding pantetheine thioester 1 (Supplementary Fig. 6a)25, which was transformed to the corresponding CoA thioester using three purified recombinant CoA biosynthetic enzymes [pantothenate kinase (PanK), phosphopantetheine adenylyl transferase (PPAT) and dephosphocoenzyme A kinase (DPCK)] and ATP as a co-substrate26. However, the CoA thioester was found to be inherently unstable in SavA enzymatic activity assays resulting in hydrolytic cleavage to the corresponding acid.

We thus turned our attention to the 10-methyldodecanoyl pantetheine thioester 1, which is a mimic of the corresponding acyl-CoA thioester. After incubation with SavA and FAD for one hour at 30°C, LC-MS analysis identified a major product with m/z=211, corresponding to [M+H]+ for the butenolide 3. Also, a pair of minor peaks with m/z=473, corresponding to [M+H]+ for the α,β-unsaturated intermediate 2, were observed (Fig. 4a). These products had very similar retention times and were absent from a control reaction employing boiled enzyme. Moreover, a time course analysis of the reaction using UV-HPLC revealed a significant increase in the level of 3 (λ=210 nm) over time, concomitant with accumulation of the putative desaturated intermediate 2 (λ=254 nm) (Fig. 4b).

To verify the structure of 3, a racemic standard was synthesized (Supplementary Fig. 6b). A Wittig reaction between 2-methylbutanal and (5-carboxypentyl) (triphenyl)phosphonium bromide yielded the unsaturated carboxylic acid, which was reduced using a combination of NaBH4 and CoCl2 to the corresponding saturated acid 7. Reduction of 7 with LiAlH4 followed by oxidation with TEMPO and PIDA yielded aldehyde 8. A modified Knoevenagel condensation of 8 with malonic acid in the presence of N-methylmorpholine afforded the β,γ-unsaturated carboxylic acid 9, which was converted to the butenolide through chlorolactonization-elimination by reaction with (COCl)2, diphenylsulfoxide, K2CO3 and 18-crown-6. The synthetic butenolide had an identical retention time to 3 from the enzymatic reaction, confirming that SavA catalyzes butenolide formation (Fig. 4a). Furthermore, 3 was isolated by semi-preparative HPLC from three 5-mL enzymatic reactions of SavA with pantetheine thioester 1 after incubation at 30°C for 10 h, and its 1H NMR spectrum was consistent with that of the synthetic standard (Supplementary Fig. 7). Meanwhile, HPLC analysis of 3 from SavA-catalyzed reaction and that of synthetic standard on a homochiral stationary phase revealed that the butenolide was produced enantioselectively (Supplementary Fig. 10a). These data confirmed that SavA is the sole enzyme responsible for homochiral butenolide formation in avenolide biosynthesis.

Dioxygen is the sole stoichiometric co-substrate of SavA
SavA catalyzes butenolide formation without requiring exogenous FAD or a separate reductant (Fig. 4a), indicating FAD’s catalytic role in this reaction is independent of external cofactors and that the enzyme may use its own substrate as electron donor.

To further examine the function of the FAD cofactor, we investigated whether the catalytic activity of SavA is enhanced by the addition of exogenous FAD. Addition of exogenous FAD did not significantly alter the overall rate of butenolide formation (Fig. 4d), consistent with the observation that SavA purifies with bound FAD, which is enough for the catalysis during the course of the reaction. After optimizing the enzymatic assay conditions, the kinetic parameters of SavA-catalyzed butenolide formation were determined (Fig. 4e), revealing a clear substrate inhibition effect of acyl pantetheine when the concentration is higher than 200 μM and moderate substrate affinity (Km=438.35±136.40 μM) and catalytic efficiency (Kcat/Km=(9.125±3.641)x10-5 μM-1S-1)).

The formation of desaturated species in early stages of the SavA-catalyzed reaction was consistent with the proposal that dehydrogenation is the first step in the process of butenolide formation, which also produces the reduced flavin required for subsequent oxygenation chemistry by SavA. To confirm this, we attempted to prepare the E- and Z-configured α,β-unsaturated pantetheine thioesters of 10-methyldodecanoic acid. (E)-10-methyldodec-2-enoyl pantetheine thioester 2 was prepared through Knoevenagel condensation of 8 with malonic acid in the presence of pyridine to give the corresponding enoic acid 10, followed by coupling with protected pantetheine and subsequent acetonide cleavage (Supplementary Fig. 6b). However, coupling of (Z)-10-methyldodec-2-enoic acid with protected pantetheine did not yield the corresponding Z-configured thioester, due to fast isomerization of the Z-configured double bond to the more stable E-isomer, promoted by the combination of thiol and base in the reaction mixture (see Supplementary Information). Comparison of synthetic (E)-2 with the products of the SavA-catalyzed reaction revealed that this shunt metabolite accumulates alongside the butenolide after prolonged periods (Fig. 4a, b). We hypothesize that the other desaturated intermediate observed in the SavA-catalyzed reaction is (Z)-2, which undergoes further oxidation to form the butenolide but can also isomerize to (E)-2 under the reaction conditions. To verify this, SavA was incubated with synthetic (E)-2. No butenolide formation was detected (Fig. 4a).

To determine the origin of the C-4 oxygen atom in butenolide 3, we incubated the enzyme with the pantetheine thioester of 10-methyldodecanoate under an 18O2 atmosphere. LC-MS analyses suggested a single 18O atom was incorporated into the product (Fig. 5a). No labeling of 3 was observed in a control reaction with air in place of 18O2. This confirmed that SavA catalyzes C-4 hydroxylation of a desaturated thioester intermediate using a reactive oxidant derived from O2. It is also in accord with the previous 18O feeding experiment showing that three of the four oxygen atoms in avenolide derive from O2, with those at C-9 and C-10 resulting from oxygen insertion reactions catalyzed by SavB (see below). Taken together, the above data lead us to propose a catalytic mechanism for SavA, involving initial desaturation of the substrate to form the corresponding Z-configured enoyl thioester and FADH2 (Fig. 5c).

It is known that ACADs and AOXs share a common reductive-half reaction in which a conserved catalytic base, typically a Glu residue23, removes the α-proton of fatty acyl-CoAs, resulting in concomitant transfer of a hydride from the β-position of the substrate to N-5 of FAD. This results in formation of an E-configured α,β-unsaturated thioester and FADH2 (Fig. 2b). In contrast, the dehydrogenation catalyzed by SavA results in formation of a Z-configured α,β-unsaturated thioester, probably due to differences in the positioning of the substrate relative to FAD and the general base in the active site of the enzyme. The FADH2 formed in this transformation reacts with O2 via single electron transfer and radical recombination to form a C4a-flavin hydroperoxide. The Z-configured α,β-unsaturated thioester then undergoes deprotonation at the γ-position by an unidentified base to yield a dienolate which reacts with the electrophilic OH of C4a-flavin hydroperoxide, resulting in cleavage of the O-O bond and γ-hydroxylation (Fig. 5c). The resulting (2Z, 4S)-4-hydroxy-2-enoyl thioester likely undergoes spontaneous cyclization with loss of pantetheine to form the butenolide. Elimination of water from the 4-hydroxyflavin by product of the hydroxylation reaction regenerates FAD for initiation of the next catalytic cycle. However, despite the well-studied flavin C4a-(hydro)peroxide oxygenation species in the reaction catalyzed by canonical flavoenzymes, alternative mechanisms cannot be excluded, such as catalytic cycle involving N5-oxygenated flavin cofactors in the form of the flavin-N5-peroxide and the flavin-N5-oxide27, or through an anionic flavosemiquinone intermediate like nitronate monooxygenase28.

To gain further insight into mechanism for butenolide formation, we aligned the sequences of several mechanistically characterized ACADs/AOXs with that of SavA and also used AlphaFold3 to predict the structure of SavA (Fig. 5b, Supplementary Fig. 8a)29. The Glu418 and Asp420 residues fall within a sequence motif located in the active site of canonical ACADs/AOXs, which typically contains a conserved Glu residue involved in α-proton abstraction. To probe the roles played by Glu418 and Asp420 in the SavA-catalyzed reaction, we created E418Q and D420N mutants of SavA. The catalytic activity of these mutants was investigated using the assay described above (Supplementary Fig. 8b). The D420N mutant was colorless, suggesting it had lost the flavin cofactor. After incubation of this mutant with pantetheine thioester 1 in the presence of FAD, UV-HPLC analyses showed that butenolide 3 is still produced, although the reaction is slower (Fig. 4c). This indicated that Asp420 does not play an essential catalytic role. However, it may be important for binding the FAD cofactor. The E418Q mutant, which was yellow, could not produce either butenolide 3 or α,β-unsaturated thioester 2, suggesting this variant is catalytically inactive, despite still being able to bind FAD (Fig. 4c). Thus, Glu418 plays a crucial role in catalysis, likely functioning as the general base responsible for removal of the α-proton in the substrate, initiating dehydrogenation (Fig. 5c). Pantetheine thioester 1 was also docked into the predicted structure of SavA to further substantiate this hypothesis. As in other ACADs, Glu418 is predicted to sit at the top of the active site pocket with the substrate positioned between this residue and the isoalloxazine ring of FAD, such that deprotonation of the α-carbon initiates the catalytic cycle (Fig. 5b).

SavB catalyzes regio/stereospecific hydroxylations of the alkyl chain
Having established that SavA assembles the butenolide moiety, we turned our attention to SavB, which was proposed to be responsible for installation of the C-9 keto and C-10 hydroxyl groups of avenolide. SavB was overproduced in E. coli as an N-terminal His6-tagged fusion protein, purified using nickel affinity chromatography and verified by UHPLC-ESI-Q-TOF-MS (Supplementary Fig. 9). Incubation of the chemically synthesized racemic butenolide 3 with SavB, NADPH, and spinach ferredoxin (Fdx)/ferredoxin reductase (FdR) at 30°C for six hours resulted in the production of avenolide, as well as two additional oxygenated species, 11 and 12, as evidenced by LC-MS analyses (Fig. 6a). Compound 11 yielded an ion with m/z=227.1641, corresponding to [M+H]+ for a hydroxylated derivative of 3, and compound 12 yielded an ion with m/z=225.1488, corresponding to [M+H]+ for a monounsaturated derivative of 11 (Fig. 6b). Moreover, a time course analysis of the SavB-catalyzed reaction revealed that 11 and 12 are intermediates in the conversion of 3 to avenolide (Fig. 6a, c).

Based on these data, we proposed that 11 is a C9-hydroxylated intermediate and 12 is its keto derivative. This was confirmed by NMR spectroscopic analyses of these compounds (Supplementary Figs. 11-16 and Supplementary Table 2) isolated by preparative HPLC from a 30 mL reaction of SavB, employing (4S)-3 isolated from the SavA-catalyzed reaction as the substrate. The frequently used CYP surrogate redox partners SelFdx1499/SelFdR0978 were used in the large-scale SavB-catalyzed reaction30. The NMR data for 12 were identical to that reported for an intermediate in the total synthesis of avenolide (Supplementary Fig. 12)31. For compound 11, a pair of signals was observed for H-9 (δ 3.55/3.48 ppm) and C-9 (δ 75.7/74.9 ppm), suggesting a mixture of syn and anti-C-9/C-10 diastereoisomers is formed (Supplementary Fig. 11). This is consistent with stereospecific monohydroxylation of C-9, because 1 was synthesized as a mixture of C-10 epimers. The stereospecific hydroxylation was also supported by HPLC analysis of 11 on a homochiral stationary phase, which only showed two peaks corresponding to a pair of diastereoisomers at C-10 (Supplementary Fig. 10b). Additionally, enzymatic assays of SavB using the isolated intermediates 11 and 12 as substrates further established that SavB catalyzes sequential, regio- and stereospecific hydroxylations of the alkyl chain first through 11 and then 12 during the conversion of 3 to avenolide (Fig. 6a, c).

Methods
Bacterial strains, plasmids and general cloning strategies
The bacterial strains and plasmids used in the study are summarized in Supplementary Table 1. The PCR was performed using Q5 high-fidelity DNA polymerase (NEB). Plasmid assembly was done by using a Uniclone One Step Seamless Cloning Kit (Genesand).

Construction of in-frame deletions in S. albidoflavus and S. avermitilis
In-frame deletion mutants of S. albidoflavus and S. avermitilis were constructed via double-crossover homologous recombination. Two DNA fragments of about 2 kb flanking the targeted regions were amplified from genomic DNA of the corresponding Streptomyces strains using the primers listed in Supplementary Data 1, and cloned into the NdeI/HindIII restriction sites of pYH7. The integrity of the resulting constructs was confirmed by sequencing and it was introduced into Streptomyces by intergeneric conjugation using ET12567/pUZ8002. The target mutant strains were screened by PCR from the apramycin-sensitive colonies prepared after two rounds of propagation on no antibiotics SFM plates. Finally, the mutant strain was reconfirmed by sequencing the PCR products.

Gene cloning
DNA fragments of savA, savB and avaR1 were individually amplified from genomic DNA of S. avermitilis MA-4680 with primers listed in Supplementary Data 1. For construction of plasmids for protein overproduction, the purified PCR products were inserted into the pET28a-A1 vector, which was linearized by NdeI and XhoI, using a Uniclone One Step Seamless Cloning Kit (Genesand) according to the manufacturer’s guidelines. For construction of the plasmid for heterologous expression of savAB, three purified PCR products containing sequences of ermE* promoter (using pOSV556t plasmid as a template), savA and savB, respectively, were cloned into the EcoRV/XbaI-digested sites of pSET152 to generate plasmid pSET152::savAB. Chemically competent E. coli DH5α cells were transformed with the cloning reaction mixture. Transformants were selected on LB agar plates supplemented with corresponding antibiotics. Plasmids were extracted from antibiotic-resistant colonies and the integrity of the cloned gene was confirmed by DNA sequencing.

Isolation and purification of avenolide from S. albidoflavus M1/pSET152::savAB
Seed culture of S. albidoflavus M1/pSET152::savAB was incubated in 250-mL Erlenmeyer flasks containing 50 mL of TSB medium with 50 µg/mL apramycin at 30°C and 220 rpm for two days, followed by inoculation into AlaMM liquid medium (2% v/v) and cultivation for seven days at the same condition. A total of 1 L fermentation was collected and centrifuged at 4568 ×g for 40 min, and the supernatant was extracted with equal volume of ethyl acetate for three times. The crude extract was separated via reverse-phase preparative HPLC equipped with a YMC-Pack ODS-A C18 column (250 × 20 mm, 5 μm), eluting with MeOH-H2O (37:63, v/v) at 7 mL/min and 210 nm to afford avenolide (tR 37.5 min). [α]D25 +67.0° (c 0.1, CH3OH); HR-ESI-MS m/z calcd for C13H21O4 [M+H]+: 241.1434 found 241.1430; 1H NMR (500 MHz, CDCl3): 7.45 (1H, dd, J=5.7, 1.4 Hz), 6.12 (1H, dd, J=5.7, 2.0 Hz), 5.05 (1H, ddt, J=8.2, 4.8, 1.8 Hz), 2.57 (1H, ddd, J=17.9, 8.0, 6.4 Hz), 2.48 (1H, ddd, J=17.9, 8.0, 6.4 Hz), 1.83 (1H, m), 1.72 (2H, q, J=7.4 Hz), 1.69 (2H, m), 1.46 (2H, m), 1.35 (3H, s), 0.80 (3H, t, J=7.4 Hz); 13C NMR (125 MHz, CDCl3): 214.3, 173.1, 156.1, 121.8, 83.1, 79.0, 35.5, 33.1, 32.5, 25.3, 24.8, 23.3, 7.8.

Protein overproduction and purification
Recombinant protein was overproduced in E. coli BL21 Star (DE3) cells carrying the corresponding plasmid. Cells were cultured in LB medium containing 50 μg/mL kanamycin at 37°C to an OD of 0.6~0.8. Expression was induced with 0.5 mM IPTG, and growth was continued at 15°C overnight. Cells were harvested by centrifugation, resuspended in loading buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mM imidazole, 10% glycerol), lysed on ice by sonication, and clarified by centrifugation at 34957 ×g at 4°C for 45 min. The supernatant containing soluble protein was filtered through a 0.45 µm filter and loaded onto a 1 mL HisTrap FF column (GE Healthcare), which had been charged with nickel and equilibrated with loading buffer. The column was washed with 10 mL loading buffer and the proteins were eluted in a stepwise manner using loading buffer containing increasing concentrations of imidazole (50, 100, 200, and 300 mM). The presence of the proteins of interest was confirmed by SDS-PAGE and fractions containing the eluted protein were combined, concentrated and buffer exchanged into storage buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10% glycerol) with Amicon centrifugal filters (Millipore). The purified protein was frozen in aliquots of 100 μL in liquid nitrogen, and stored at -80°C. Protein concentrations were measured by BCA Protein Assay (Thermo) according to the manufacturer’s instructions.

Site-directed mutagenesis
The E418Q and D420N mutants of His6-SavA were created by PCR-based site-directed mutagenesis using the pET28a-A1-savA plasmid as a template. The primers used are listed in Supplementary Data 1. The constructs were sequenced to confirm the presence of the desired mutations. Recombinant SavA mutants were overproduced and purified by the procedures described above. SDS-PAGE analysis of the mutants is shown in Supplementary Fig. 8.

Electrophoretic mobility shift assay
Part of the intergenic region between avaR1 and savA (191 bp) containing the proposed binding site for AvaR1 were amplified from genomic DNA of S. avermitilis MA-4680 with primers listed in Supplementary Data 1. The EMSA was run on a 6% non-denaturing polyacrylamide gel. Lane 1 contained 0.024 pmol of isolated DNA only and lanes 2-7 contained 0.024 pmol of DNA and 4.0 pmol of AvaR1. For lanes 3 to 7, increasing concentrations of avenolide (dissolved in DMSO) were added (0, 0.5, 5, 12.5 and 25 nmol respectively). Each 20 μL reaction mixture contained 4 μL of 5× binding buffer (100 mM HEPES, pH 7.6, 150 mM KCl, 50 mM (NH₄)₂SO₄, 5 mM DTT, 5 mM EDTA, 1% (w/v) Tween 20). DNA, protein, binding buffer and distilled water were combined and incubated at room temperature for 15 min. Following the addition of the compound solution, the mixture was further incubated at room temperature for 10 min. Then, 5 μL of loading buffer (0.25× TBE, 34% (v/v) glycerol, 0.2% (w/v) bromophenol blue) was added, bringing the total reaction volume to no more than 25 μL. Samples were resolved on a pre-run native polyacrylamide gel in 1× TBE buffer at 80 V and 4°C until the loading dye reached the bottom of the gel. After electrophoresis, the gel was stained with gentle agitation in 0.005% (v/v) GelRed prepared in 1× TBE for 30 min at room temperature, and then visualized using a UV transilluminator.

UV-Vis spectroscopic analysis and confirmation of FAD cofactor of SavA
SavA was first confirmed as a flavoprotein by UV-Vis spectroscopic analysis. 200 µL of purified SavA (100 µM) was prepared in a buffer containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl in a brown quartz cuvette, and the reduced SavA (100 µM) was obtained by mixing SavA with sodium dithionite (2 mM) and subsequent reaction at room temperature for 20 min in quartz cuvette. The samples were analyzed by UV-Vis spectroscopy using a Shimadzu UV-2550 UV/VIS spectrophotometer scanned between 200 and 800 nm. An equal volume of the buffer and FAD (50 µM) were used as controls.

The cofactor was released by incubating SavA (100 μM) at 100°C for 10 min in the dark. Denatured protein was removed by centrifugation at 13680 ×g at 4°C for 10 min. The supernatant was filtered and subjected to UV-HPLC analysis with a C-18 column (150 × 4.6 mm, 5 μm, 30°C). The column was eluted with a linear gradient of 5–30% MeCN containing 0.1% formic acid at a flow rate of 1 mL/min for 30 min. The retention time of released cofactor from SavA was identical with that of authentic FAD.

In vitro enzymatic assay of SavA and its mutants
SavA (50 μM) was incubated with synthetic 10-methyldodecanoyl pantetheine 1 (1.0 mM) in a total volume of 100 μL, with or without FAD (100 μM). Reactions were carried out in a buffer containing 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 10% glycerol at 30°C for 1 h. The reactions were quenched by adding equal amount of ethyl acetate and extracted for three times, and the combined organic layers were concentrated in vacuo. The residue was dissolved in 100 μL MeOH and analyzed by UV-HPLC and LC-MS. In the time-course experiment of SavA, reactions were incubated at 30°C and quenched at different timepoints. For the assay of SavA mutants, pantetheine thioester 1 (1.0 mM) was incubated with SavA mutants (50 μM) in the presence of FAD (100 μM) at 30°C for 3 h.

To determine the kinetic parameters of the SavA-catalyzed reaction, 10-methyldodecanoyl pantetheine 1 was set at the concentration of 10, 20, 30, 40, 60, 80, 90, 100, 120, 140, 170, 200, 250, 300, 350, 500, and 700 μM. Enzyme assays were performed in 20 mM Tris-HCl pH 8.0, 100 mM NaCl containing 10 μM SavA and 20 μM FAD at 30°C for 10 min in triplicate. The reactions were quenched by adding an equal volume of MeOH. After centrifugation, the supernatant was subjected to HPLC analyses. Kinetic parameters (Km, Kcat, Vmax and Ki) were calculated by nonlinear regression analysis of substrate concentration versus initial velocity plots using Substrate Inhib function in the Origin 2024 software.

Large-scale reaction of SavA and isolation of butenolide 3
To obtain the butenolide for NMR characterization, 3 × 5 mL reactions were carried out. To a stirred solution of pantetheine thioester 1 (2.5 mM) and FAD (50 μM) in 5 mL of buffer containing 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 10% glycerol was added SavA (59 μM). The reactions were stirred at 30°C and consumption of the substrate was monitored by UV-HPLC at wavelengths λ=210 nm and 254 nm. After reaction for 10 h, the separate parallel reactions were combined and extracted with equal amount of ethyl acetate for three times. The organic layers were concentrated in vacuo and the residue was purified over a YMC-C18 semi-preparative column eluted with 80% MeOH as mobile phase at a flow rate of 3 mL/min to afford butenolide 3. HR-ESI-MS m/z calcd for C13H23O2 [M+H]+: 211.1693 found 211.1696. The 1H and 13C NMR data of 3 are summarized in Supplementary Table 2.

SavA-catalyzed incorporation of 18O2 into butenolide 3
To determine the origin of the ring oxygen atom of butenolide 3, 18O2 was carefully bubbled into a solution of SavA (47 μM) and FAD (100 μM) in a total volume of 100 μL buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl) for 1 min to replace the air. After addition of a solution of pantetheine thioester 1 (100 mM stock, 1 μL), the Eppendorf tube was sealed immediately and the resulting mixture was incubated at 30°C for 1 h. The reaction was extracted with 200 μL ethyl acetate for three times, and the combined organic layers were concentrated in vacuo. The residue was dissolved in 100 μL MeOH and analyzed by LC-MS.

In vitro enzymatic assay of SavB
Partially purified ferredoxin (1.0–3.0 mg/mL solution in 0.15 M Trizma buffer, pH 7.5 and NaCl) and ferredoxin reductase (8.5 units made to 0.5 mL in Tris 25 mM, pH 8, 0.1 M NaCl, 20% glycerol) from Spinacia oleracea were purchased from Sigma and were used without further purification. Purified SavB (9 μM) was incubated with NADPH (1.0 mM), ferredoxin (2 μL), ferredoxin reductase (2 μL) and the chemically synthesized racemic butenolide 3 (1.0 mM, stock dissolved in DMSO) in a total volume of 100 μL of buffer containing 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 10% glycerol. The reaction was incubated at 30°C for 6 h. The reaction was then extracted with 200 μL ethyl acetate for three times, and the combined organic layers were concentrated in vacuo. The residue was dissolved in 100 μL MeOH and analyzed by UV-HPLC and LC-MS. In the time-course experiment of SavB, reactions were incubated at 30°C and quenched at different timepoints. In vitro enzymatic assays of SavB with intermediates 11 and 12 were conducted following the same procedure except that the reaction was incubated for 2 h before being quenched.

Large-scale reaction of SavB and isolation of intermediates 11 and 12
To obtain the intermediates 11 and 12 for NMR characterization, a 30-mL reaction was carried out. Purified SavB (25 μM) was incubated with NADPH (4.0 mM), SelFdx1499 (30 μM), SelFdR0978 (12 μM) and the isolated butenolide 3 (1.3 mM) from the large-scale SavA reaction in a total volume of 30 mL of buffer containing 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 10% glycerol. The reaction was stirred at 30°C and consumption of the substrate was monitored by UV-HPLC at wavelength λ=210 nm. After incubation for 3.5 h, the reaction was extracted with 20 mL ethyl acetate for four times and the organic layers were concentrated in vacuo and the residue was purified over a YMC-C18 semi-preparative column eluted with 65% MeOH as mobile phase at a flow rate of 3 mL/min to afford 11 (2.4 mg, tR = 12.3 min, HR-ESI-MS m/z calcd for C13H23O3 [M+H]+: 227.1642 found 227.1641), and 12 (2.7 mg, tR = 9.9 min, HR-ESI-MS m/z calcd for C13H21O3 [M+H]+: 225.1485 found 225.1488). The 1H and 13C NMR data of 11 and 12 are summarized in Supplementary Table 2.

UPLC-ESI-Q-TOF-MS analyses of intact proteins
UPLC-ESI-Q-TOF-MS analyses of protein samples were performed on a Waters Acquity UPLC H-Class Plus System equipped with a BEH C4 130Å reverse phase column (1.7 μm, 2.1 × 50 mm) coupled to a SYNAPT XS mass spectrometer with a standard ESI mode [positive mode; capillary voltage, 3.0 kV; cone voltage, 40 V, source heating temperature, 120 °C, desolvation temperature, 600°C; nebulizer gas (N2), 6.5 bar; cone gas flow, 100 L/hr; desolvation gas flow, 600 L/hr; m/z range 400–3000; scan time 0.2 sec]. Mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Chromatographic separation was achieved using a linear gradient starting from 3% B to 97% B in 5 min at a flow rate of 0.2 mL/min and 80°C column temperature. Lock mass data was collected during each acquisition and the correction was applied during post acquisition data processing via MassLynx Software v4.2. MaxEnt1 in UNIFI software v3.9.0.23 by Waters was used for deconvolution of mass spectra.

UV-HPLC and LC-MS analyses of enzymatic products
For analyzing the enzymatic products, UV-HPLC experiments were performed using a Shimadzu Prominence-i LC-2030 Plus HPLC system equipped with a Welch Xtimate C18 reverse-phase column (5 μm, 4.6 mm × 200 mm). The column was eluted with solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) using the following elution profile at a flow rate of 1.0 mL/min: 30% B for 3 min; linear gradient from 30% B to 100% B over 35 min; 100% B for 5 min; linear gradient from 100% B to 30% B over 1 min; 30% B for 6 min. Labsolution by Shimadzu was used to collect and analyze UV/Vis data.

For chiral HPLC analyses of 3 and 11, UV-HPLC experiments were performed using a Shimadzu Prominence-i LC-2030 Plus HPLC system equipped with a Daicel CHIRALPAK AY-3R column (3 μm, 4.6 mm × 250 mm). Chiral separation of compound 3 was performed under isocratic elution with 70% acetonitrile (containing 0.1% formic acid in water) at a flow rate of 0.5 mL/min. The analysis of compound 11 was performed under isocratic elution with 60% acetonitrile (containing 0.1% formic acid in water) at a flow rate of 0.5 mL/min.

LC-MS analyses were performed using an Agilent 1200 HPLC system equipped with a Sinochrom ODS-BP column (5 μm, 4.6 mm × 200 mm) coupled to an Agilent Accurate-Mass-Q-TOF 6520 mass spectrometer in positive ion mode [mass range, 80–2000 m/z; drying gas, 300°C, 11 L/min; nebulizer, 45 psi; capillary voltage, 3500 V; fragmentor, 130 V; skimmer, 65 V; octupole RF Peak, 750 V]. The samples were analyzed using the above elution profile at a flow rate of 1.0 mL/min. LC-MS data were collected using MassHunter Qualitative Analysis B.05.01 software and analyzed using MassHunter Qualitative Analysis B.06.00 software.

Molecular docking
Docking study was performed within the Schrödinger Maestro software package (version 12.8.117) based on the united atom scoring function. The structure of pantetheine thioester 1 was prepared using the LigPrep program from Schrödinger’s Maestro software suite and the OPLS4 force field was utilized. Possible protonation and ionization states were assigned using Epik at pH 7.0 ± 0.2. Possible stereoisomers and tautomeric states were generated. The predicted three-dimensional structure of SavA-FAD complex from AlphaFold3 was prepared using the Protein Preparation Wizard in the Schrödinger Maestro software suite by adding hydrogens. PROPKA (Schrödinger) was used to adjust the charges and protonation states (at pH 7.0). Structure was optimized and the energy-minimized using the OPLS4 force field. Docking stimulations were performed using the Glide program in Schrödinger with the receptor grid prepared using the OPLS4 force field. The grid center was set to be the centroid of Glu418, and the side length of the cubic grid was set to 20 Å. No other constraints were used in the receptor grids. Flexible ligand sampling with the SP (Standard Precision) docking mode was utilized, followed by post docking minimization to generate a maximum of ten ligand poses. All other parameters were set to defaults for the Glide docking process.

Synthesis of the substrate and product standards for enzymatic assays
The procedures used and the compound characterization data obtained are detailed in the Supplementary Information.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Li et al. report the elucidation of the biosynthesis of avenolide, a hormone that regulates avermectins production in\nStreptomyces avermitilis. Through heterologous expression and in vitro assays, they showed that the trifunctional\nflavoenzyme SavA catalyzes a unique desaturation and hydroxylation–lactonization cascade to form a homochiral 4-\nalkylbutenolide, and SavB, a cytochrome P450, then installs three sequential, regio- and stereospecific hydroxylations, to\ncomplete avenolide assembly. This work highlights a sustainable biocatalytic route for butenolide synthesis using dioxygen\nas the sole stoichiometric reagent. The authors successfully synthesized the fatty acid acyl-phosphopantetheine (Ppant)\nsubstrate for SavA using a chemo enzymatic method. Furthermore, by comparing the product with a synthesized standard\nand employing 18O2 as a labeled substrate, they identified the reaction of SavA acting on acyl carrier protein. They also\nidentified a key residue E418 which abstracts a proton from the substrate, based on the docking model by using the\nAlphaFold3 structure of SavA. Therefore, the quality of the biochemical analysis in this study can be considered high. The\nsequential oxidations catalyzed by SavA and SavB are intriguing as a point view of enzymology. New insights into the\nbiosynthesis of butyrolactone will contribute to elucidating its diversity as a signaling molecule and secondary metabolite,\nmaking this research highly relevant to the fields of biosynthesis, chemical biology, and microbiology. Thus, the reviewer\nrecommends this study for publication after the authors corrected minor points as described below.\n1. (Figure 4a) What is the peak eluted at 28.3 min? The authors should label the product by using the product number.\n2. (Line 314) The authors should cite the Figure S10-11 here.\n3. (Line 419) The calculated ms should be described here.\n4. The reviewer thinks that the additional discussion on the catalysis by SavA should help the readers to understand the\nuniqueness of its catalysis. This reviewer recommends the authors to compare the catalysis of canonical ACAD/AOX\nenzymes with those of SavA and EpnF.\n5. The preference of SavA on the Ppant tethered substrates sound unique. Are there any ACAD/AOX enzymes which accept\nPpant-tethered substrates? The authors should describe the structural basis which accommodate Ppant arm or carrier\nprotein based on the docking model.\n6. Do the avenolide intermediates obtained in this study possibly work as a signaling molecule? If possible, the author\nshould do electrophoretic mobility shift assay for AvaR by using them.\n7. The tables for the NMR assignments of 3, 11 and 12 should be presented.", "reviewer_2": "The manuscript by Li, Zhou and colleagues reports the biosynthesis of the actinobacterial transcriptional regulator avenolide\ncontaining a butanolide moiety. By combining heterologous expression, gene inactivation, chemical synthesis and in vitro\nenzyme assays, the authors convincingly demonstrate the involvement of two oxidoreductases, i.e. flavoenzyme SavA that\nproduces the butenolide ring from a medium-chain fatty acyl thioester, followed by CypP40 SavB that completes avenolide\nformation by several oxygenation reactions. 18O-isotope labelling is employed to support the incorporation of a total of 3\noxygen atoms from O2 into the substrate. Sustainable routes for biocatalytic asymmetric butanolide formation are of\nindustrial interest and this study identified two enzymes that could potentially be applied. Overall, the data supporting the\nrole of these two enzymes is convincing and verifies an enzymatic route independent of the canonical PBS. Currently, the\nproposed SavA mechanism is plausible but not further supported with mechanistic or structural data and the enzymes are\nnot characterized in terms of their kinetic properties and overall substrate turnover, which would be relevant when\nconsidered as potential (starting point for the development of) green catalysts. Here, especially the CYP SavB seemed to\nshow very low turnover and activity. Nonetheless, this is an interesting study that is very well presented and pleasant to\nread. A few points should be considered prior to publication:\nIn my opinion, the field of flavoenzyme biochemistry could be better introduced and/or discussed. For example, I suggest to\nbriefly mention the current classification of flavoprotein monooxygenases = FPMOs (e.g. PMID: 33588053) and the\ndistinction between “external” (requiring exogenous e-donors such as NADPH) and “internal” (using the substrate as e-\ndonor) FPMOs, SavA belonging to the latter. Group D FPMOs have an ACAD fold but are typically two-component external\nFPMOs (i.e. the flavin is reduced by a separate flavin reductase and then taken up by the oxygenase component), so I guess\nSavA is not a classical group D FPMO (same for EpnF and TdaE - an enzyme involved in the biosynthesis of bacterial\ntropone natural products). These enzyme rather seem to represent some new type of internal FPMO which may evolutionary\nderive from former ACADs and combine typical ACAD chemistry (dehydrogenation of a substrate) with FPMO chemistry (O2\nreduction and substrate oxygenation).\nEnzyme mechanism: The proposed mechanism is plausible but it is not a given that C4a-peroxide species are used by\nthese ACADs. For TdaE, N5-oxygenated flavins (N5-peroxide, N5-oxide) appear more likely (PMID: 34196542), which have\nalso been reported before in other internal FPMOs from Streptomyces involved in natural product biosynthesis. It is even\npossible that no covalent flavin-oxygen species are formed, similar to nitronate monooxygenase (PMID: 19577534). Without\nfurther evidence (e.g. stopped-flow spectroscopy to verify a C4a-peroxide, quenching to confirm transient N5-oxide species\nby MS etc.), I suggest to at least briefly point out alternative mechanisms in the text and adjust the mechanistic discussion.\nAlso in that regard, EpnF and TdaE function as epoxidases rather than hydroxylases, which may possibly also be the case\nfor SavA.\nIt could also be interesting to construct a phylogenetic tree of typical ACADs as well as EpnF, TdaE and SavA (and possible\nadditional enzymes) and compare active site features of the three internal FPMOs, not sure though if that will give any\ninsights....\nCurrently, no turnover numbers, kinetics etc. are shown in the paper. The assays should allow to determine some basic\nkinetics, at least for SavA (SavB seems to be very slow, maybe not surprisingly for a CYP lacking native e-donors), e.g. KM,\nvmax, catalytic efficiency?\nMinor points:\nFig. 3c: Why is there so much unlabeled product with m/z of 241? Under 18O2 atmosphere one would expect more than that,\ncf. also to Fig. 5a, where >80% seem to be labelled?\nL176: Protein is not homogeneous after Ni2+ affinity column based on Fig. 5a. Lots of impurities.\nL222: “Exogenous FAD is not required for SavA to catalyze butenolide formation and no reductant capable of regenerating\nFAD from FADH2 is included in the enzymatic reaction mixture, suggesting FAD plays a catalytic role”. Please rephrase\n(FAD can also play a catalytic role when NADPH is required), e.g., to “SavA catalyzes butenolide formation without requiring\nexogenous FAD or a separate reductant, indicating FAD's catalytic role in this reaction is independent of external cofactors\nand that the enzyme functions as internal FPMO using its own substrate as electron donor.”\nAlso the statement “…observation that SavA purifies with bound FAD, which is not consumed during the course of the\nreaction” is somewhat imprecise. What do the authors mean exactly by “consumed”? FAD is normally never “consumed”\n(used up) in flavoenzymes and typically restored to FAD after each reaction cycle. For SavA, FAD is first reduced to FADH2\nby the substrate and then reoxidized with O2 to generate the putative oxygenating species for subsequent oxygen transfer to\nthe substrate (in contrast to oxidases, which would simply reduce O2 to H2O2).\nFig S5: For spectra in panel c, please adjust y- and x-axes (absorption <300 not relevant) so that the distinct flavin spectral\nproperties are highlighted. Also in panel d: “authetic” typo and FMN should also be shown as standard for comparison (or\nMS data verifying FAD).\nFig. 4b: I suggest to directly indicate time in min in this figure instead of using (i), (ii) and (iii).\n“The formation of desaturated species in early stages of the SavA-catalyzed reaction was consistent with the proposal that\ndehydrogenation is the first step in the process of butenolide formation.” This is to some extent a necessity because the\nreaction order cannot be inverted when the electrons for flavin reduction and O2 activation come from the substrate, consider\nadjusting to “The formation of desaturated species in early stages of the SavA-catalyzed reaction was consistent with the\nproposal that dehydrogenation is the first step in the process of butenolide formation, which also produces the reduced flavin\nrequired for subsequent oxygenation chemistry by SavA.”", "reviewer_3": "This manuscript presents a compelling study on the enzymatic biosynthesis of avenolide, a 4-alkylbutenolide hormone\ninvolved in regulating avermectin production in Streptomyces avermitilis. The authors elegantly demonstrate that SavA, a\nmultifunctional flavoenzyme, catalyzes a remarkable desaturation-hydroxylation-lactonization cascade to form the\nhomochiral butenolide core, utilizing dioxygen as the sole stoichiometric reagent. The subsequent tailoring by SavB, a\ncytochrome P450, introduces regio- and stereospecific hydroxylations to complete avenolide assembly. The experimental\ndesign is robust, integrating in vitro enzymatic assays, isotopic labeling, and mutagenesis studies to substantiate the\nproposed mechanism. The writing is clear and accessible, making the work readily digestible for a broad audience. The\ndiscovery of SavA’s unique multifunctionality and its potential as a biocatalyst for sustainable asymmetric butenolide\nsynthesis represents a significant advance in the field. This work is highly novel and merits publication pending minor\nrevisions addressing the following points:\nSpecific Comments:\n1) Identification of Intermediate E/Z Isomers and Hydroxylation Regiochemistry:\nIn the SavA in vitro assay (Fig. 4a), two m/z peaks corresponding to compound 2 were detected. The authors assigned one\npeak as the E-isomer based on comparison with a synthetic standard. The other peak may correspond to the Z-isomer, but\nthis remains speculative since the Z-isomer was not tested as a substrate for SavA. Alternatively, competitive hydroxylation\nat C-3 vs. C-4 could occur:\nIf C-3 hydroxylation occurs, dehydration would yield E-2, which may not proceed to form 3.\nIf C-4 hydroxylation occurs, lactonization (possibly via nucleophilic attack on the thioester) followed by desaturation could\nyield the α,β-unsaturated lactone.\nTo clarify the mechanism, we recommend probing the regiochemistry and order of hydroxylation steps, e.g., by testing\npossible intermediates and/or conducting additional isotopic labeling.\n2) Stereochemical Assignment and Catalytic Efficiency of SavB:\nThe configuration of the C-4 hydroxy group in compound 3 (generated by SavA) was assigned by comparison with a\nracemic synthetic standard. However, this does not confirm whether the stereochemistry matches that of the final product (1).\nTo unequivocally establish enantiopurity, the authors should use enantiomerically pure substrates or products for\ncomparison.\nIn the SavB in vitro assay (Fig. 6a), the use of racemic 3 as substrate makes it difficult to conclude whether SavB is\nstereoselective. It is essential to use enantiopure 3 to assess stereospecificity and catalytic efficiency.\nFig. 6a shows that the synthetic standard of 1 contains a small amount of 3. Could this impurity affect the interpretation of the\nassay? Additionally, SavB’s catalytic efficiency appears low. Have the authors explored alternative redox partners or\ncofactors (e.g., different ferredoxins/ferredoxin reductases) to optimize activity? The low efficiency may be due to suboptimal\npartner proteins or reaction conditions.\n3) Order of Oxidations Catalyzed by SavB:\nThe proposed sequence of C-9 and C-10 oxidation (C-9 hydroxylation → dehydrogenation/dihydroxylation-dehydration →\nC-10 hydroxylation) is intriguing but relies largely on inference. To rigorously establish the order, the authors could test\nputative intermediates (e.g., compounds 11 and 12, as mentioned) in SavB in vitro assays. This would provide direct\nevidence for the oxidative cascade and strengthen the mechanistic proposal.\n4）Substrate Scope and Versatility of SavA:\nThe SavA-catalyzed cascade (desaturation, hydroxylation, lactonization) is highly intriguing. To enhance the general\napplicability and impact of this study, it would be valuable to test whether SavA can accept alkyl substrates of varying chain\nlengths to produce butenolides with different alkyl substituents. If such versatility is demonstrated, it would significantly\nbroaden the enzyme’s utility for biocatalytic applications. If not, please discuss the potential for enzyme engineering to alter\nsubstrate specificity or analyze structural/mechanistic constraints that may limit substrate flexibility.\nConclusion:\nThis work is of high quality and represents an important contribution to the fields of enzymatic catalysis. The mechanistic\ninsights into SavA’s multifunctionality are particularly noteworthy. Addressing the above points will further elevate the impact\nand rigor of the study. Thus, I recommend minor revisions."}, "author_response": "We thank the referee for appreciating the quality and significance of the work we have\nundertaken.\n1. (Figure 4a) What is the peak eluted at 28.3 min? The authors should label the\nproduct by using the product number.\nWe speculate that the peak eluted at 28.3 min is the desaturated intermediate (Z)-2,\nwhich undergoes further oxidation to form butenolide 3 but can also isomerize to (E)-\n2 under the reaction conditions. We have labelled the peaks by using the product\nnumber in Fig. 4a and added an explanatory note to the legend in the revised\nmanuscript.\n2. (Line 314) The authors should cite the Figure S10-11 here.\nWe have cited the corresponding Supplementary Figures here (Figures S11-16 and\nTable S3 in the revised manuscript).\n3. (Line 419) The calculated ms should be described here.\nThe calculated MS data has been added here in the revised manuscript.\n4. The reviewer thinks that the additional discussion on the catalysis by SavA should\nhelp the readers to understand the uniqueness of its catalysis. This reviewer\nrecommends the authors to compare the catalysis of canonical ACAD/AOX enzymes\nwith those of SavA and EpnF.\nMany thanks for this suggestion. An additional figure (Supplementary Fig. 17) has been\nincluded in the revised SI that compares the catalysis of canonical ACAD/AOX\nenzymes with SavA, EpnF, and TdaE, an enzyme involved in the biosynthesis of\nbacterial tropone natural products as mentioned by reviewer #2. Concise discussion\nhas also been added in the revised manuscript.\nAs this figure shows, canonical ACADs/AOXs only introduce α,β-trans double bonds\ninto fatty acid, while SavA catalyzes a unique desaturation-hydroxylation-lactonization\ncascade and EpnF catalyzes a decarboxylation-desaturation-epoxidation cascade. In\nthe case of TdaE, a complex cascade reaction comprising dehydrogenation,\nnoncannonical CoA ester oxygenolysis and epoxidation was involved.\n5. The preference of SavA on the Ppant tethered substrates sound unique. Are there\nany ACAD/AOX enzymes which accept Ppant-tethered substrates? The authors\nshould describe the structural basis which accommodate Ppant arm or carrier protein\nbased on the docking model.\nACADs/AOX enzymes normally accept fatty acid acyl CoA of varying chain lengths as\nsubstrates to introduce α,β-desaturation during each cycle of fatty acid β-oxidation in\nperoxisomes or mitochondria. There is ample precedent for the structure of these\nenzymes complexed with Ppant-tethered substrates, mostly acyl CoA (e.g., Proc. Natl.\nAcad. Sci. U.S.A. 2020, 117, 16324).\nIn the docking model of SavA with acyl panthetheine thioester 1, the pantetheine arm\nof the substrate connects the fatty acid chain to the catalytic site of the enzyme and\nlocate the substrate at an appropriate position between E418 and FAD, which is similar\nto canonical ACAD/AOXs. To clarify the interactions, we have updated Fig. 5b and\nadded more information in the legend. However, as there is no actual structural\ninformation available, we prefer not to speculate much at this stage.\n6. Do the avenolide intermediates obtained in this study possibly work as a signaling\nmolecule? If possible, the author should do electrophoretic mobility shift assay for\nAvaR by using them.\nThe avenolide intermediates obtained in the study include the butenolide 3, 9-\nhydroxylated intermediate 11 and 10-deoxy avenolide 12. Among them, the last\nintermediate in the biosynthetic pathway, 10-deoxy avenolide, was synthesized and\ntested in EMSA experiments with AvaR1 in previous work about the discovery and total\nsynthesis of avenolide (Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 16410 and J. Antibiot.\n2011, 64, 781). However, it has been reported that even the 10-deoxy form showed\nsignificantly reduced activity compared to avenolide (1.2 : 100), indicating that the\ncomplete structure of avenolide is essential for its biological function as a signaling\nmolecule. Therefore, we think these intermediates are unlikely to work as signaling\nmolecules.\n7. The tables for the NMR assignments of 3, 11 and 12 should be presented.\nThanks to the reviewer for this suggestion. A table summarizing the NMR assignments\nof compounds 3, 11 and 12 has been added as Supplementary Table 3 in the revised\nSI.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Li, Zhou and colleagues reports the biosynthesis of the\nactinobacterial transcriptional regulator avenolide containing a butanolide moiety. By\ncombining heterologous expression, gene inactivation, chemical synthesis and in vitro\nenzyme assays, the authors convincingly demonstrate the involvement of two\noxidoreductases, i.e. flavoenzyme SavA that produces the butenolide ring from a\nmedium-chain fatty acyl thioester, followed by CypP40 SavB that completes avenolide\nformation by several oxygenation reactions. 18O-isotope labelling is employed to\nsupport the incorporation of a total of 3 oxygen atoms from O2 into the substrate. 18O-isotope labelling is employed to\nsupport the incorporation of a total of 3 oxygen atoms from O2 into the substrate. Sustainable routes for biocatalytic asymmetric butanolide formation are of\nindustrial interest and this study identified two enzymes that could potentially be applied. Overall,\nthe data supporting the role of these two enzymes is convincing and verifies an\nenzymatic route independent of the canonical PBS. Currently, the proposed SavA\nmechanism is plausible but not further supported with mechanistic or structural data\nand the enzymes are not characterized in terms of their kinetic properties and overall\nsubstrate turnover, which would be relevant when considered as potential (starting\npoint for the development of) green catalysts. Here, especially the CYP SavB seemed\nto show very low turnover and activity.\nNonetheless, this is an interesting study that is very well presented and pleasant to\nread. A few points should be considered prior to publication:\nWe are grateful to the reviewer for recognizing the quality of the work we have\nundertaken.\nIn my opinion, the field of flavoenzyme biochemistry could be better introduced and/or\ndiscussed. For example, I suggest to briefly mention the current classification of\nflavoprotein monooxygenases = FPMOs (e.g. PMID: 33588053) and the distinction\nbetween “external” (requiring exogenous e-donors such as NADPH) and “internal”\n(using the substrate as e-donor) FPMOs, SavA belonging to the latter. Group D FPMOs\nhave an ACAD fold but are typically two-component external FPMOs (i.e. the flavin is\nreduced by a separate flavin reductase and then taken up by the oxygenase\ncomponent), so I guess SavA is not a classical group D FPMO (same for EpnF and\nTdaE - an enzyme involved in the biosynthesis of bacterial tropone natural products).\nThese enzyme rather seem to represent some new type of internal FPMO which may\nevolutionary derive from former ACADs and combine typical ACAD chemistry\n(dehydrogenation of a substrate) with FPMO chemistry (O2 reduction and substrate\noxygenation).\nMany thanks to the reviewer for this suggestion. We have included more information\nin the conclusions section in the revised manuscript, which introduces the current\nclassification of FPMOs and highlights the unusual functionality of SavA. An additional\nfigure (Supplementary Fig. 17) has been included in the revised SI that highlights the\ncatalysis of canonical ACAD/AOX enzymes with SavA, EpnF and TdaE. Additional\nreferences have also been added to the main manuscript.\nEnzyme mechanism: The proposed mechanism is plausible but it is not a given that\nC4a-peroxide species are used by these ACADs. For TdaE, N5-oxygenated flavins\n(N5-peroxide, N5-oxide) appear more likely (PMID: 34196542), which have also been\nreported before in other internal FPMOs from Streptomyces involved in natural product\nbiosynthesis. It is even possible that no covalent flavin-oxygen species are formed,\nsimilar to nitronate monooxygenase (PMID: 19577534). Without further evidence (e.g.\nstopped-flow spectroscopy to verify a C4a-peroxide, quenching to confirm transient\nN5-oxide species by MS etc.), I suggest to at least briefly point out alternative\nmechanisms in the text and adjust the mechanistic discussion. Also in that regard,\nEpnF and TdaE function as epoxidases rather than hydroxylases, which may possibly\nalso be the case for SavA.\nMany thanks for this suggestion. We have adjusted the mechanistic discussion and\nalso pointed out alternative mechanisms in the legend of Fig. 5c in the revised\nmanuscript. We understand that further evidence would be needed to elucidate the\nprecise mechanism underlying SavA’s catalysis and these would be interesting\nexperiments to do in future.\nIt could also be interesting to construct a phylogenetic tree of typical ACADs as well as\nEpnF, TdaE and SavA (and possible additional enzymes) and compare active site\nfeatures of the three internal FPMOs, not sure though if that will give any insights....\nMany thanks for the suggestion. Some typical ACADs/AOXs from various species and\nSavA homologues which presumably share the same catalytic function as SavA, e.g.,\nOrf18 proposed to catalyze the synthesis of avenolide-type signaling molecule SFB1\nwhich regulates the production of tylosin in Streptomyces fradiae (Appl. Microbiol.\nBiotechnol. 2025, 109, 18) and SsnA identified from Streptomyces sp. NHF165 as a\nbiosynthetic enzyme for avenolide-like signaling molecule (unpublished data), were\naligned with these internal FPMOs. The phylogenetic tree has been constructed using\nmaximum-likelihood method (see below). However, no significant similarity between\nSavA and other internal FPMOs was found both in sequence or active site, implying\nSavA represents a new type of internal FPMOs.\nCurrently, no turnover numbers, kinetics etc. are shown in the paper. The assays\nshould allow to determine some basic kinetics, at least for SavA (SavB seems to be\nvery slow, maybe not surprisingly for a CYP lacking native e-donors), e.g. KM,\nvmax, catalytic efficiency?\nThe relevant kinetics data of SavA, including K , V and catalytic efficiency, has been\nm max\nadded as Fig. 4e in the revised manuscript. More information has also been added in\nthe figure legend and the corresponding results and methods section.\nMinor points:\nFig. 3c: Why is there so much unlabeled product with m/z of 241? Under 18O\n2\natmosphere one would expect more than that, cf. also to Fig. 5a, where >80% seem\nto be labelled?\nThe mass spectrum in Fig. 5a was derived from LC-MS analysis of a small volume\n(100 μL) of SavA enzyme reaction in a 1.5 mL Eppendorf tube, which was bubbled with\n18O gas to replace the air. However, the mass spectrum in Fig. 3c was derived from\nwith 18O gas and grown in a 100 mL container for seven days. Strictly speaking, the\n2\nlatter culture was grown under a mixture of normal air and 18O , due to the bigger\n2\nvolume of the container and difficulty in replacing air with 18O .\nproduct with m/z of 247 clearly indicates that up to three 18O atoms were incorporated\ninto avenolide structure, in comparison to that observed in the control experiment using\nair.\nL176: Protein is not homogeneous after Ni2+ affinity column based on Fig. 5a. Lots of\nimpurities.\nThe recombinant SavA protein was efficiently overproduced and purified, obtained in\nhigh yield. The corresponding SDS-PAGE analysis has been updated to a more\nrepresentative version in Supplementary Fig. 5 for improved clarity.\nL222: “Exogenous FAD is not required for SavA to catalyze butenolide formation and\nno reductant capable of regenerating FAD from FADH2 is included in the enzymatic\nreaction mixture, suggesting FAD plays a catalytic role”. Please rephrase (FAD can\nalso play a catalytic role when NADPH is required), e.g., to “SavA catalyzes butenolide\nformation without requiring exogenous FAD or a separate reductant, indicating FAD's\ncatalytic role in this reaction is independent of external cofactors and that the enzyme\nfunctions as internal FPMO using its own substrate as electron donor.”\nAlso the statement “…observation that SavA purifies with bound FAD, which is not\nconsumed during the course of the reaction” is somewhat imprecise. What do the\nauthors mean exactly by “consumed”? FAD is normally never “consumed” (used up) in\nflavoenzymes and typically restored to FAD after each reaction cycle. For SavA, FAD\nis first reduced to FADH2 by the substrate and then reoxidized with O2 to generate the\nputative oxygenating species for subsequent oxygen transfer to the substrate (in\ncontrast to oxidases, which would simply reduce O2 to H2O2).\nMany thanks for the suggestion. We have rephrased the paragraph here to make it\nmore precise and better fit the context.\nFig S5: For spectra in panel c, please adjust y- and x-axes (absorption <300 not\nrelevant) so that the distinct flavin spectral properties are highlighted. Also in panel d:\n“authetic” typo and FMN should also be shown as standard for comparison (or MS data\nverifying FAD).\nMany thanks for the suggestions and drawing our attention to the typographical error.\nThe Supplementary Fig. 5 has been updated in the revised SI. And the MS data\nverifying FAD has been added as Fig. S5e.\nFig. 4b: I suggest to directly indicate time in min in this figure instead of using (i),\n(ii) and (iii).\nThe Fig. 4b has been updated as suggested in the revised manuscript.\n“The formation of desaturated species in early stages of the SavA-catalyzed reaction\nwas consistent with the proposal that dehydrogenation is the first step in the process\nof butenolide formation.” This is to some extent a necessity because the reaction order\ncannot be inverted when the electrons for flavin reduction and O2 activation come from\nthe substrate, consider adjusting to “The formation of desaturated species in early\nstages of the SavA-catalyzed reaction was consistent with the proposal that\ndehydrogenation is the first step in the process of butenolide formation, which also\nproduces the reduced flavin required for subsequent oxygenation chemistry by SavA.”\nMany thanks for the suggestion. This sentence has been adjusted in the revised\nmanuscript.\nReviewer #3 (Remarks to the Author):\nThis manuscript presents a compelling study on the enzymatic biosynthesis of\navenolide, a 4-alkylbutenolide hormone involved in regulating avermectin production\nin Streptomyces avermitilis. The authors elegantly demonstrate that SavA, a\nmultifunctional flavoenzyme, catalyzes a remarkable desaturation-hydroxylation-\nlactonization cascade to form the homochiral butenolide core, utilizing dioxygen as the\nsole stoichiometric reagent. The subsequent tailoring by SavB, a cytochrome P450,\nintroduces regio- and stereospecific hydroxylations to complete avenolide assembly.\nThe experimental design is robust, integrating in vitro enzymatic assays, isotopic\nlabeling, and mutagenesis studies to substantiate the proposed mechanism. The\nwriting is clear and accessible, making the work readily digestible for a broad audience.\nThe discovery of SavA’s unique multifunctionality and its potential as a biocatalyst for\nsustainable asymmetric butenolide synthesis represents a significant advance in the\nfield. This work is highly novel and merits publication pending minor revisions\naddressing the following points:\nWe are grateful to the referee for recognizing the quality and novelty of the work we\nhave undertaken.\nSpecific Comments:\n1) Identification of Intermediate E/Z Isomers and Hydroxylation Regiochemistry:\nIn the SavA in vitro assay (Fig. 4a), two m/z peaks corresponding to compound 2 were\ndetected. The authors assigned one peak as the E-isomer based on comparison with\na synthetic standard. The other peak may correspond to the Z-isomer, but this remains\nspeculative since the Z-isomer was not tested as a substrate for SavA. Alternatively,\ncompetitive hydroxylation at C-3 vs. C-4 could occur:\nIf C-3 hydroxylation occurs, dehydration would yield E-2, which may not proceed to\nform 3.\nIf C-4 hydroxylation occurs, lactonization (possibly via nucleophilic attack on the\nthioester) followed by desaturation could yield the α,β-unsaturated lactone.\nTo clarify the mechanism, we recommend probing the regiochemistry and order of\nhydroxylation steps, e.g., by testing possible intermediates and/or conducting\nadditional isotopic labeling.\nWe agree there is still no direct evidence confirming that the other desaturated\nintermediate observed in the SavA-catalyzed reaction is (Z)-2. Despite extensive\nsynthetic efforts, the isomerization of the Z-configured double bond to the more stable\nE-isomer proceeds rapidly, promoted by the thiol and base in the reaction mixture,\nmaking preparation of the Z-configured thioester unachievable. This is also in accord\nwith the fact that E-isomer is present in the SavA-catalyzed reaction, which may be\nisomerized from Z-2 under the enzymatic reaction conditions.\nHowever, the proposal of the dehydrogenation-hydroxylation-lactonization cascade for\nSavA is most consistent with the properties of such flavin-dependent enzymes.\nThe absence of a requirement for exogenous electron donors (e.g., NADPH) makes it\nunlikely that hydroxylation is the first step in the SavA-catalyzed reaction. Current\nevidence from in vitro enzymatic reaction supports that SavA is more like an internal\nflavoprotein monooxygenase which uses the substrate as electron donor.\nWe therefore propose that dehydrogenation is the initial step in butenolide formation,\nthereby generating the reduced flavin required for subsequent oxygenation by SavA.\nThis was also agreed by reviewer #2 (This is to some extent a necessity because the\nreaction order cannot be inverted when the electrons for flavin reduction and O\n2\nactivation come from the substrate).\n2) Stereochemical Assignment and Catalytic Efficiency of SavB:\nThe configuration of the C-4 hydroxy group in compound 3 (generated by SavA) was\nassigned by comparison with a racemic synthetic standard. However, this does not\nconfirm whether the stereochemistry matches that of the final product (1). To\nunequivocally establish enantiopurity, the authors should use enantiomerically pure\nsubstrates or products for comparison.\nMany thanks for this suggestion. To clarify this, we have conducted HPLC analysis of\ncompound 3 that was isolated from a larger-scale enzymatic reactions of SavA with\nthe pantetheine thioester 1, and the racemic synthetic 3 on a homochiral stationary\nphase. The enzymatic product only gave a pair of peaks corresponding to the\nasymmetric hydroxylated product at C-4, while the synthetic standard showed four\npeaks corresponding to the presence of four stereoisomers. The results clearly indicate\nthat SavA catalyzes homochiral 4-alkylbutenolide formation. The HPLC chromatogram\nhas been added as Supplementary Fig. 10a in the revised SI.\nIn the SavB in vitro assay (Fig. 6a), the use of racemic 3 as substrate makes it difficult\nto conclude whether SavB is stereoselective. It is essential to use enantiopure 3 to\nassess stereospecificity and catalytic efficiency.\nMany thanks for the suggestion. We agree that enantiopure 3 would be better for the\nstudy, however, its preparation would be much more complex and costly. To\ncomplement current data for the stereochemical assignment of SavB-catalyzed\nproducts, HPLC analysis of intermediate 11 (isolated from a large-scale SavB reaction\nusing (4S)-3 from SavA reaction as the substate) on a homochiral stationary phase\nhas been conducted and only a pair of peaks were observed, which unambiguously\nindicates that SavB indeed catalyzes a stereoselective hydroxylation. Together with\nthe clear stereochemistry of avenolide reported, we thus conclude that SavB’s\ncatalysis is a stereoselective process.\nWe have revised the description in the corresponding paragraph in the revised\nmanuscript and added the HPLC chromatogram in Supplementary Fig. 10b.\nFig. 6a shows that the synthetic standard of 1 contains a small amount of 3. Could this\nimpurity affect the interpretation of the assay? Additionally, SavB’s catalytic efficiency\nappears low. Have the authors explored alternative redox partners or cofactors (e.g.,\ndifferent ferredoxins/ferredoxin reductases) to optimize activity? The low efficiency\nmay be due to suboptimal partner proteins or reaction conditions.\nThe peak having a same retention time as compound 3 in avenolide standard in Fig.\n6a corresponds to an unknown impurity, but not 3, which has been confirmed in LC-\nMS analysis. To avoid the misinterpretation, the avenolide standard was re-analyzed\nand UV chromatograms have been updated in Fig. 6a in the revised manuscript.\nYes, different redox partners have been tried in the SavA enzyme assays, such as the\nfrequently used spinach Fdx/Fdr, SelFdx1499/SelFdR0978 and others. However, most\nof them didn’t give better results than the spinach Fdx/FdR pair. The low efficiency of\nSavB is very likely due to suboptimal partner proteins, which was also noted by\nreviewer #2 (SavB seems to be very slow, maybe not surprisingly for a CYP lacking\nnative e-donors).\n3) Order of Oxidations Catalyzed by SavB:\nThe proposed sequence of C-9 and C-10 oxidation (C-9 hydroxylation →\ndehydrogenation/dihydroxylation-dehydration → C-10 hydroxylation) is intriguing but\nrelies largely on inference. To rigorously establish the order, the authors could test\nputative intermediates (e.g., compounds 11 and 12, as mentioned) in SavB in vitro\nassays. This would provide direct evidence for the oxidative cascade and strengthen\nthe mechanistic proposal.\nMany thanks for this suggestion. The SavB enzymatic assays using compounds 11\nand 12 as substrates were conducted and the results further supported the proposed\nsequence of reactions. More information has been added in the corresponding\nparagraph and UV chromatograms from in vitro assays have been added in Fig. 6a in\nthe revised manuscript.\n4）Substrate Scope and Versatility of SavA:\nThe SavA-catalyzed cascade (desaturation, hydroxylation, lactonization) is highly\nintriguing. To enhance the general applicability and impact of this study, it would be\nvaluable to test whether SavA can accept alkyl substrates of varying chain lengths to\nproduce butenolides with different alkyl substituents. If such versatility is demonstrated,\nit would significantly broaden the enzyme’s utility for biocatalytic applications. If not,\nplease discuss the potential for enzyme engineering to alter substrate specificity or\nanalyze structural/mechanistic constraints that may limit substrate flexibility.\nMany thanks for the suggestion. The discovery of the unique catalytic function of SavA\nlays the foundation for development of sustainable biocatalytic asymmetric butenolide\nsynthesis, which is also the focus of our current study. The substrate scope and\nversatility of SavA, as well as the structural basis for its catalysis, is very important for\nthe following biocatalytic applications. These would be the focus of our work in the next\nstep.\nConclusion:\nThis work is of high quality and represents an important contribution to the fields of\nenzymatic catalysis. The mechanistic insights into SavA’s multifunctionality are\nparticularly noteworthy. Addressing the above points will further elevate the impact and\nrigor of the study. Thus, I recommend minor revisions.\nWe thank the reviewer for appreciating the quality and significance of the work we have\nundertaken, and for the above valuable suggestions that have helped us to improve\nthe manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In this revision, the authors have adequately addressed all of the concerns raised by this reviewer as well as the other two\nreviewers. I now recommend publication of this work without any further changes.", "reviewer_2": "The revised manuscript by Li, Zhou and colleagues has been thoroughly revised and is much improved in my opinion. All\nthe points I raised were properly addressed and I have no further suggestions.", "reviewer_3": "The revised manuscript is acceptable."}, "author_response": ""}]

Decision letter