Engineering LmrR protein for L-proline-based asymmetric aldol biocatalysis

Domain: chemistry
Published: 2026-02-23
Editorial outcome: minor_revision

Abstract
L-Proline is a powerful organocatalyst widely applied in asymmetric synthesis due to its secondary amine functionality. However, in proteins, this functional group is locked in peptide bonds, rendering proline catalytically inactive. Natural enzymes that leverage L-proline-based catalysis are exceedingly rare. Here, we engineer the nonenzymatic protein scaffold LmrR into a new-to-nature biocatalyst by exposing its native L-proline residue at the N-terminus to catalyze enantioselective aldol reactions. Through rational design, protein engineering, and reaction optimization, we develop an engineered LmrR variant that achieves up to 99% conversion and >99% enantiomeric excess across a range of aromatic and heteroaromatic aldehyde substrates. Our findings reveal a unique strategy for unlocking dormant catalytic potential in natural amino acids and protein scaffolds, providing an applicable approach to create tailored, L-proline-based enzymes for asymmetric synthesis.

Introduction
Enzymes offer green and sustainable solutions for synthetic chemistry due to their high enantioselectivity, rapid reaction rates, and operation under environmentally benign conditions1-3. Consequently, they have been widely employed in the synthesis of various valuable molecules from pharmaceutical building blocks to complex natural products4-6. However, natural enzymes are inherently constrained by their evolved biological functions, which often fall short of meeting the diverse and growing demands of industrial and pharmaceutical synthesis. To overcome these limitations, extensive efforts have focused on enzyme engineering7,8, directed evolution9,10, and chemical modification11,12 to enhance catalytic selectivity, efficiency, and substrate scope.

The design and evolution of artificial enzymes have emerged as powerful strategies to expand the catalytic capabilities of proteins beyond those found in nature13,14. Several nonenzymatic protein scaffolds (e.g., streptavidin, serum albumins, and multidrug resistance regulators) have been repurposed through the incorporation of noncanonical catalytic elements, resulting in artificial enzymes with novel reactivities, improved stability, and enhanced catalytic efficiency15-17. Among these, the bacterial transcriptional regulator LmrR from Lactococcus lactis has attracted attention as a versatile scaffold due to its large hydrophobic pocket at the dimer interface, which facilitates substrate binding18,19. The Roelfes group and others have extensively engineered LmrR into functional biocatalysts, including metalloenzymes and photoenzymes, for a range of abiological chemical transformations20-27. Notably, a boronic acid-dependent LmrR-based enzyme was developed to catalyze enantioselective organocatalytic reactions inaccessible to natural enzymes25. In another example, a synthetic triplet photosensitizer was incorporated into LmrR to enable photoenzymatic energy transfer cycloadditions24. These advances underscore the versatility and evolvability of LmrR. However, such designs typically rely on genetic code expansion—incorporating noncanonical amino acids via amber suppression—to create functional catalytic sites20-27. This technique remains limited by the availability of orthogonal translation systems, high costs of noncanonical amino acids, misincorporation, and reduced protein expression yields16. To date, no artificial enzyme based on LmrR has utilized only native residues as catalytic sites.

L-Proline and its derivatives are widely used in asymmetric organic synthesis, enabling key transformations such as aldol reactions28,29, Michael additions30,31, and Mannich reactions32,33, among others34-37. The catalytic activity of L-proline arises from its secondary amine, which forms an enamine intermediate with carbonyl substrates36. However, in proteins, this functional group is typically inactivated by peptide bond formation, making proline-based catalysis extremely rare in nature. One of the few known exceptions is 4-oxalocrotonate tautomerase (4-OT), which possesses an N-terminal L-proline that mediates secondary amine catalysis and has been engineered for asymmetric reactions38-44. Inspired by this precedent, we aim to exploit the N-terminal L-proline of LmrR as a native catalytic element, avoiding the need for noncanonical amino acids or synthetic cofactors.

Here, we engineer the nonenzymatic transcriptional regulator LmrR into a natural aldolase-like enzyme by exposing its native L-proline residue at the N-terminus to serve as an organocatalyst (Fig. 1). To achieve this, we reposition the proline residue in LmrR from position five to the N-terminus, thereby liberating its secondary amine within the hydrophobic pocket to enable enantioselective aldol catalysis. Specifically, we modify the LmrR sequence by removing the first four residues and subsequently deleting three additional residues to shift the exposed N-terminal L-proline deeper into the hydrophobic pocket (Fig. 1b). Through rational design and reaction optimization, we obtain an LmrR variant that mimics natural aldolase activity, exhibiting high catalytic efficiency in asymmetric aldol reactions with substrate conversions up to 99% and enantiomeric excess (e.e.) values over 99% (Fig. 1c). This work presents a rational strategy for designing new-to-nature biocatalysts from the nonenzymatic protein scaffold (LmrR). While demonstrated successfully with LmrR, this enzyme design strategy depends on the presence and position of proline residues within the scaffold protein. In cases where a suitable proline is absent, a proline residue could be directly introduced at the N-terminus and subsequently repositioned into a hydrophobic cavity to achieve catalysis and stereoselective chemical transformations.

Results
Discovery of LmrR-catalyzed aldol reaction
LmrR has been widely employed as a privileged scaffold for the design of artificial enzymes, owing to its large hydrophobic pocket that can accommodate a broad range of substrates20-27. Although LmrR lacks any known native enzymatic function14,19,25, its hydrophobic pocket alone may nonetheless support latent catalytic activity. To probe this possibility, we selected a model asymmetric aldol reaction between cyclohexanone and 4-nitrobenzaldehyde and evaluated its efficiency in the presence of LmrR (Fig. 2a). Remarkably, LmrR (1.5 mol%) catalyzed the reaction with 95% conversion (based on 20 mM 4-nitrobenzaldehyde), albeit with low enantioselectivity (7% e.e.) (Fig. 2b). For comparison, we assessed L-proline (1.5 mol%) under the same conditions. Yet, no product formation was observed in either aqueous or organic media (Fig. 2b), consistent with the requirement for high catalyst loadings (typically up to 30 mol%) in conventional proline-catalyzed aldol reactions28 (Fig. 1a). These results suggest that LmrR provides a catalytic advantage over free L-proline, likely due to substrate preorganization within its hydrophobic pocket, and highlight the potential of native LmrR as a starting point for enzyme-like aldol catalysis.

Encouraged by this unexpected catalytic activity, we next investigated which residues might contribute to the native aldolase-like activity of LmrR. Sequence analysis revealed that LmrR contains nine lysine residues (Fig. 1b; see the full sequence in Supplementary Information). Given that lysine’s ε-amino group (primary amine) is essential for catalysis in class I aldolases45-48, we hypothesized that these residues could be mediating the observed aldol reaction. To verify this hypothesis, we first mutated three lysine residues (K6, K22, and K101), located near the hydrophobic pocket, to alanine, generating the LmEK variant (Fig. 1b). This mutation significantly reduced the conversion to ~10%. However, further mutating the remaining six lysine residues to alanine (yielding LmrR_No Lys; with K77 substituted by glutamine to improve solubility) resulted in only a slightly additional decrease in activity (~8% conversion) (Fig. 2b). To further rule out contributions from primary amine groups at the protein surface or N-terminus, we chemically blocked all primary amines using a t-butyloxycarbonyl (Boc) protecting group. The Boc-modified LmrR and LmrR_No Lys showed similar conversion rates to LmEK, without further reduction (Fig. 2b). Together, these results suggest that the three lysine residues (K6, K22, and K101) proximal to the active site primarily account for LmrR’s intrinsic catalytic activity.

To further validate the role of the three lysine residues in catalysis, we conducted reductive amination-based trapping experiments to probe their interaction with the aldol substrates. Specifically, parental LmrR and its lysine-to-alanine mutant (LmEK) were incubated with cyclohexanone, 4-nitrobenzaldehyde, and sodium triacetoxyborohydride [NaBH(OAc)3], a mild reductant that irreversibly traps transient enamine intermediates via covalent bond formation (Fig. 2c). Following incubation, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis revealed a mass shift in parental LmrR consistent with three covalent modifications—corresponding to the addition of three cyclohexane-derived groups (Fig. 2d, top). In contrast, no such modification was observed in the LmEK variant (Fig. 2d, bottom), confirming the involvement of K6, K22, and K101 in forming the enamine intermediate. These data demonstrate that the three lysine residues proximal to the hydrophobic pocket provide nucleophilic primary amines essential for the observed aldol reactivity of LmrR, achieving up to 95% substrate conversion (Fig. 2b). We next evaluated LmrR variants retaining one or two lysine residues in the hydrophobic pocket. All such variants showed high conversions (93-96%) but low enantioselectivities (6-11% e.e.) (Supplementary Fig. 1). These findings indicate that although lysine residues (K6, K22, K101) can serve as catalytic nucleophiles, the LmrR pocket does not provide the chiral environment necessary for effective stereocontrol. While natural lysine-dependent aldolases achieve high enantioselectivity through highly organized active sites48-50, engineering LmrR to create a similarly preorganized chiral pocket around lysine would require extensive structural redesign. Therefore, in this work we pursued an alternative and complementary strategy, leveraging proline-mediated enamine catalysis to introduce a chiral catalytic element directly into the active site.

Rational design of LmrR for high enantioselectivity
With the LmrR variant LmEK (Fig. 1b) in hand, we aimed to enhance its enantioselectivity through rational design and protein engineering (Fig. 3a). Our approach consisted of three main strategies. First, leveraging the broad catalytic versatility of L-proline’s secondary amine28-37, we repositioned the native proline residue at position 5 to the N-terminus of LmEK by deleting the initial four residues (G1–I4), thereby exposing the proline as a free N-terminal organocatalyst. This design mimics the rare natural enzyme 4-oxalocrotonate tautomerase (4-OT), which features an N-terminal L-proline as its catalytic center38-44. Second, to optimize substrate binding, we further truncated residues following the N-terminal proline to relocate it closer to the hydrophobic pocket’s core, where two central tryptophan residues (W96 and W96’ in native LmrR) provide π–π interactions to recruit aromatic substrates19,20,24. Third, we applied targeted mutagenesis to polar residues surrounding the proline in the pocket, aiming to reduce unfavorable hydrogen bonding with the substrates that might hinder effective catalysis. Based on precedent from 4-OT38,51 and classical proline organocatalysis52-54, we propose that our engineered LmrR catalyzes the aldol reaction via an enamine intermediate formed between the secondary amine of the N-terminal proline and cyclohexanone (Fig. 3b), facilitating nucleophilic addition to the aldehyde.

To initiate our design, we deleted the first four residues (G1–I4) from both LmEK and parental LmrR, generating the variants LPEK1 and LNP, respectively. Since these proteins were expressed in E. coli, endogenous methionine aminopeptidase catalyzed the cleavage of the N-terminal methionine from the nascent polypeptides55-57, thereby exposing the L-proline residue at position 1. The successful removal of the N-terminal methionine in all variants was confirmed by MALDI-TOF-MS, which showed excellent agreement between the observed and calculated molecular weights (Fig. 3c). To confirm that the exposed N-terminal L-proline mediated the proposed enamine catalysis (Fig. 3b), we repeated the reductive trapping experiments by incubating the variants with the substrates and NaBH(OAc)3. Notably, the LNP variant derived from parental LmrR exhibited a major mass peak corresponding to four cyclohexane modifications (Fig. 3c), indicating that the N-terminal proline trapped one substrate molecule in addition to the three lysine residues (K6, K22, and K101) previously identified (Fig. 2d, top panel). Upon mutation of these three lysines to alanine, all four LPEK variants showed only a single modification peak, consistent with trapping exclusively by the N-terminal L-proline residue (Fig. 3c and Supplementary Fig. 2). Collectively, these results confirm that in our engineered LPEK variants, the N-terminal L-proline functions as the sole organocatalyst mediating enamine-based catalysis.

Next, we evaluated the catalytic performance of various LmrR variants. Notably, all variants, including parental LmrR, exhibited similarly high conversions ranging from 88% to 96% (Fig. 3d), demonstrating the robustness of these enzymes for this transformation. However, their enantioselectivities varied significantly (Fig. 3e). Exposing the L-proline residue at the N-terminus (variant LNP) resulted in a more than threefold increase in enantioselectivity compared to parental LmrR, highlighting the pivotal role of L-proline in asymmetric catalysis. Further improvement was observed in LPEK1, where mutation of the three lysine residues (K6, K22, and K101) in the hydrophobic pocket enhanced enantioselectivity. Building on this, starting from LPEK1—which exhibited moderate enantioselectivity (34% e.e.; Fig. 3e)—we conducted three rounds of protein engineering to further boost enantioselectivity (Fig. 1b). In the first round, we sought to reposition the N-terminal proline (P1) closer to the center of the hydrophobic pocket by deleting one, two, or three residues following P1 (Fig. 3a, bottom). Deleting all three residues (A2, E3, and M4) to generate the LPEK2 variant led to an approximately 40% increase in enantioselectivity relative to LPEK1 (Fig. 3e). Structural comparisons revealed that the distance between P1 and residue W89’ (formerly W96’ in parental LmrR) was shorter in LPEK2 than in LPEK1 (Supplementary Fig. 3), consistent with our hypothesis that shifting P1 closer to the pocket center enhances enantiocontrol.

Based on LPEK2, we initiated the second round of protein engineering by targeting residues near P1, specifically polar amino acids Q5, R49, S88, S90, and D93, for mutation. These residues were substituted with nonpolar amino acids of similar steric bulk (Fig. 1b), aiming to disrupt hydrogen bonding with the substrate and thereby enhance enantioface discrimination. Among the resulting variants, LPEK2_S88A (designated LPEK3) exhibited the most significant improvement, achieving an enantioselectivity of 66% e.e. (Fig. 3e). Molecular docking suggests this improvement arises from the elimination of a hydrogen bond between the hydroxyl group (-OH) of S88 and the nitro group (-NO2) of 4-nitrobenzaldehyde (Supplementary Fig. 4a). Recognizing the critical role of residue S88 in enantioselectivity, we performed saturation mutagenesis at this position. While several S88 mutants showed enhanced enantioselectivity compared to LPEK2, none surpassed the performance of LPEK3 (LPEK2_S88A; Supplementary Fig. 4b).

In the third round, we aimed to increase the flexibility of P1 within the hydrophobic pocket by disrupting the N-terminal rigid α-helix. To achieve this, we targeted residues R3 and Q5 for mutation to glycine (G) and/or proline (P). Glycine’s small side chain confers high flexibility, while proline’s unique cyclic structure disrupts α-helix formation by preventing hydrogen bonding. Using LPEK3 as the parent, we generated variants and evaluated their catalytic performance. Among them, the variant LPEK3_R3P_Q5G (designated LPEK4) showed the highest enantioselectivity, reaching 73% e.e. (Fig. 3e). These results highlight the advantage of introducing localized structural disorder to increase flexibility (Supplementary Fig. 5), a factor that may be beneficial for enhancing protein functions such as enantioselectivity in our designer enzymes. To examine whether an N-terminal primary amine could also mediate stereoselective catalysis, we constructed LPEK4_P1A, in which the N-terminal proline was replaced with alanine. Although LPEK4_P1A retained high catalytic activity (92% conversion), its enantioselectivity remained low (11% e.e.) (Supplementary Fig. 6). These results indicate that the N-terminal secondary amine of proline is essential for stereocontrol in the LPEK variants. Taken together, through rational design and protein engineering, we improved the enantioselectivity of the designer enzyme LPEK4 (73% e.e.) to more than tenfold that of the initial parental LmrR (7% e.e.).

Finally, we characterized the structure and kinetics of the optimized variant LPEK4. We employed AlphaFold prediction, circular dichroism (CD) spectroscopy, size exclusion chromatography (SEC), and transmission electron microscopy (TEM) to assess structural integrity. Our analyses showed that all variants maintained well-preserved structures with characteristics consistent with parental LmrR (Supplementary Figs. 7-10). Despite the deletion of a short N-terminal fragment comprising seven residues, each variant retained its hydrophobic pocket, supporting high catalytic activity and substrate conversion (Fig. 3d). We also measured kinetic parameters (kcat and Km) for LPEK4 and LmrR (Supplementary Fig. 11). Although the catalytic efficiency of LPEK4 (kcat/Km = 1.14 × 10-2 mM-1 s-1) was lower than that of LmrR (kcat/Km = 2.59 × 10-2 mM-1 s-1), LPEK4 exhibited a markedly improved enantioselectivity compared to the parental enzyme (Fig. 3e). In addition, we measured the kinetic parameters of three LmrR variants (LmEK_A6K, LmEK_A22K, and LmEK_A101K), each retaining only a single lysine residue in the active site (Supplementary Fig. 12). These variants also exhibited substantially reduced catalytic efficiencies (0.59-0.86 x 10-2 mM-1 s-1) compared to LmrR containing three lysine residues. Together with the kinetic data for LPEK4, these observations suggest that the reduced catalytic efficiency arises primarily from the reduced number of catalytically active residues in the pocket, rather than from the nature of the N-terminal proline itself. Yet importantly, this reduction in activity is accompanied by a substantial gain in enantioselectivity, highlighting the trade-off between turnover and stereochemical control.

Optimization of LPEK4-based catalysis
Using the optimal LPEK4 variant, we next optimized the reaction conditions for the asymmetric aldol addition, focusing on two key parameters (reaction temperature and pH) that influence catalytic activity and enantioselectivity of enzymes58-61. We first varied the reaction temperature from 10 to 30 °C in PBS buffer (pH 7.3) with 1.5 mol% biocatalyst. The results indicated that the lowest temperature (10 °C) favored enantioselectivity, achieving 81% e.e. compared to 71% e.e. at 30 °C, although the substrate conversion was reduced at the lower temperature (Fig. 4a). This trend aligns with previous studies showing that temperature can modulate enzyme enantioselectivity58-60.

Next, we fixed the temperature at 10 °C and examined the effect of pH on LPEK4 performance. Because the conversion after 24 h at 10 °C was relatively low (63%), we extended the reaction time to 48 h. Under these conditions, conversion exceeded 95% at pH values above 5.8 (Fig. 4b), but decreased to about 60% at more acidic pH levels of 5.0 and 3.0. Notably, the highest enantioselectivity (90% e.e.) was observed at pH 5.8. Overall, a mildly acidic environment (pH 5.0-6.6) enhanced enantioselectivity (85-90% e.e.) compared to more acidic or neutral pH conditions (Fig. 4b). In addition, the pKa of the P1 residue in LPEK4 was experimentally determined to be 5.6 (Supplementary Fig. 13), consistent with its predicted value (pKa 5.5) obtained using H++62,63. At the reaction pH (5.8), P1 is therefore predominantly in the deprotonated form, facilitating enamine formation. By comparison, P1 in LPEK1 has a higher predicted pKa (7.0), indicating that repositioning P1 into the hydrophobic pocket of LPEK4 is essential for lowering its pKa and achieving effective catalysis. Taken together, the optimal conditions for LPEK4-catalyzed asymmetric aldol addition were determined to be 10 °C, pH 5.8, and a reaction time of 48 h, resulting in up to 95% conversion and 90% enantioselectivity.

Substrate scope of LPEK4
Finally, we investigated the substrate tolerance of our designer enzyme LPEK4 by catalyzing cyclohexanone (1) with various 4-nitrobenzaldehyde analogs (2a-l) bearing different substituents on the phenyl or heteroaromatic ring (Fig. 4c). All reactions were performed under the optimized conditions, and substrate conversion (based on aldehyde 2), e.e. value, and diastereomeric ratio (d.r.) were analyzed. The results showed that LPEK4 efficiently tolerated a range of substituents, including NO2 (2a-c), CN (2d,e), Cl (2f,g), and COOMe (2h,i) on the phenyl ring, achieving excellent conversions of 89-99%. Notably, enantioselectivity and diastereoselectivity were highest when the substituents were located at the para-position relative to the aldehyde group, followed by those at the meta- and ortho-positions (3a-i). For example, the product 3h, derived from a para-COOMe substituent, was obtained with 93% conversion and excellent levels of enantioselectivity (>99% e.e.) and diastereoselectivity (94:6 d.r.). This trend likely reflects that para-substituents are spatially distant from both the reactive aldehyde and catalytic residues (e.g., the N-terminal L-proline), minimizing steric or electronic interference with the secondary amine-mediated asymmetric aldol reaction. Furthermore, when the phenyl ring of 2 was replaced with a pyridine ring (2j-l), LPEK4 maintained good catalytic efficiency and selectivity, producing 3j-l with up to 81% conversion and 91% e.e.. Together, these results demonstrate the robustness and broad substrate promiscuity of LPEK4, which is consistent with previous reports on other LmrR-based designer enzymes such as photoenzymes24, metalloenzymes27, and boron-dependent biocatalysts25.

Methods
Expression and purification of designer enzymes
LmrR and its derived designer enzymes were expressed in Escherichia coli BL21 Gold cells transformed with a pET17b-based plasmid encoding the respective gene. Seed cultures were grown overnight at 37 °C in 20 mL of Luria–Bertani (LB) medium. Subsequently, 15 mL of the seed culture was used to inoculate 1 L of fresh LB medium in a 2.5-L flask, followed by incubation at 37 °C. When OD600 reached 0.5-0.6, protein expression was induced with 0.5 mM IPTG, and cells were further cultivated at 20 °C for 16 h.

Cells were harvested by centrifugation at 5000 g and 4 °C for 10 min, then washed once with lysis buffer (10 mM Tris-HCl, 200 mM NaCl, pH 7.0). The cell pellets were resuspended in lysis buffer and lysed by sonication (50% amplitude, 10 s on/off for a total of 40 min). The cell lysate was clarified by centrifugation at 21000 g and 4 °C for 20 min. The resulting supernatant was filtered through a 0.45 μm membrane and subjected to purification using a 5-mL Ni-NTA column (GE Healthcare). Eluted proteins were desalted and concentrated using Amicon Ultra-15 centrifugal filters (3 kDa cutoff, Merck) by centrifugation at 4000 g and 4 °C. Protein concentrations were determined by measuring absorbance at 280 nm using a UV-Vis micro-spectrophotometer (Nano-300, Hangzhou Allsheng Instruments, China). Typical protein yields were between 30 and 50 mg/mL. Purified proteins were flash-frozen in liquid nitrogen and stored at −80 °C until use.

General procedure for enzyme-catalyzed reactions
Enzymatic reactions were performed in a total volume of 50 μL in 2 mL microcentrifuge tubes, using purified enzymes and substrates. A standard reaction mixture consisted of 0.3 mM enzyme, 200 mM cyclohexanone, and 20 mM 4-nitrobenzaldehyde in 10 mM PBS buffer (pH 7.3). Reactions were incubated at 30 °C for 12 hours with shaking at 600 rpm (Thermo-Shaker, MSC-300, Hangzhou Allsheng Instruments, China). Upon completion, each reaction was quenched with 450 μL acetonitrile and analyzed by HPLC to determine substrate conversion. For determination of e.e. values, products were extracted with a tenfold volume of ethyl acetate and then analyzed by chiral HPLC.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors report the redesign of the nonenzymatic protein scaffold LmrR into a non-natural enantioselective aldolase that uses an N-terminal Pro-1 residue as the key catalytic residue. The engineering of L-proline-based enzymes for asymmetric C-C bond formation (including enantioselective aldol reactions) has already been achieved by Poelarends and coworkers with 4-oxalocrotonate tautomerase (4-OT). However, I do not think this diminishes the impact since 4-OT is a truly exceptional protein, so achieving this with a different nonenzymatic protein is not necessarily expected. Furthermore, the applied design strategy is interesting and unique. The authors engineered the LmrR sequence by removing the first four residues, exposing its native L-proline residue at the N-terminus, and subsequently deleting three additional residues to shift the exposed N-terminal L-proline deeper into the hydrophobic pocket. This was followed by directed evolution and optimization of reaction conditions to improve performance. The authors explored the substrate scope using cyclohexanone and several benzaldehyde derivatives, demonstrating that the enzyme can be used to produce a range of enantioenriched aldol products.\nThis work is interesting and original in terms of the applied enzyme design strategy, but the following comments need to be addressed before publication:\nThe authors mention that there findings reveal a unique strategy to create tailored, L-proline based enzymes for asymmetric synthesis. The authors should clearly mention that their strategy appears to be limited to protein sequences with a proline residue close to the N-terminus, which can also be relocated into a hydrophobic pocket. Alternatively, a Pro-1 residue can be directly introduced at the N-terminus, followed by repositioning into a hydrophobic cavity.\nThe authors have demonstrated that the three lysine residues proximal to the active site primarily account for LmrR’s intrinsic aldolase activity. Why did the authors not use K6 as the key catalytic residue to evolve an artificial aldolase? This seems much simpler than the applied strategy of relocating Pro-5 to the N-terminus, followed by placing it deeper into LmrR’s hydrophobic pocket? The authors should discuss both potential strategies. Also, the argument that a proline residue (secondary amine) would be better to achieve high stereoselectivity compared to a lysine residue (primary amine) perhaps makes sense for organocatalysis, but not necessarily for enzymatic reactions. Note that the chiral environment of the active site largely determines stereocontrol, as shown by the highly enantioselective aldol reactions catalyzed by many lysine-based aldolases. This should be more carefully discussed as well.\nThe authors claim that in the engineered LPEK1–LPEK4 enzymes, the N-terminal L-proline functions as the sole ‘organocatalyst’ mediating enamine-based catalysis, indicating that the secondary amine of the Pro-1 residue is crucial for the improved performance. In the best variant (LPEK4), the authors should make the Pro-1 to Ala-1 mutation to demonstrate that indeed the secondary amine of Pro-1 is required rather than an N-terminal primary amine (as present in Ala-1). Would a primary amine group work equilly well? This is an important point to experimentally address because the authors claim a unique engineering strategy to develop L-proline-based enzymes, with a catalytically competent secondary amine, for asymmetric transformations.\nTo optimize the LPEK4-catalyzed aldol reactions, optimal reaction conditions were determined. How does the selected pH of 5.8 relate to the pKa value of the Pro-1 residue? It would be informative to determine the pKa of the pro-1 residue. The authors should also make clear in the mansuscript that the placement of the Pro-1 residue in a hydrophobic pocket is crucial to lower its pKa value.\nThroughout the manuscript the authors mention that the artificial aldolase catalyzes an aldol condensation reaction. I believe it is more appropriate to call it an aldol addition reaction, as no dehydration of the aldol product takes place.\nP2/3: “One of the few known exceptions is 4-oxalocrotonate tautomerase (4-OT), which possesses an N-terminal L-proline that mediates secondary amine catalysis and has been engineered for asymmetric reactions38-41.” Here the authors should be more accurate and include the following references on 4-OT catalyzed asymmetric transformations (Angew Chem Int Ed Engl. 2022 Feb 14;61(8):e202113970. doi: 10.1002/anie.202113970; Angew Chem Int Ed Engl. 2021 Nov 2;60(45):24059-24063; Angew Chem Int Ed Engl. 2020 Jun 22;59(26):10374-10378. doi: 10.1002/anie.202001373).\nP11: “Given the broad utility of L-proline-based organocatalysis in asymmetric synthesis….” Here the authors should also mention that there is a large body of literature showing the broad utility of L-proline-based biocatalysts in asymmetric synthesis (work on 4-OT by Poelarends and coworkers).\nFrom the SI it is not clear how racemic and enantiopure reference compounds have been prepared? This seemingly missing information should be added to the SI. It is also not clear how the absolute configuration has been determined? Spectra with appropriate references seem to be missing and should be added for at least a few products.", "reviewer_2": "The manuscript by Lu et al. presents an interesting example of engineering the LmrR protein scaffold to feature an N-terminal L-proline residue capable of participating in enamine-based aldol catalysis. The central innovation lies in repositioning the N-terminus so that a canonical L-proline becomes catalytically exposed, eliminating the need for non-canonical amino acids or post-translational modifications to introduce a secondary amine. The authors further demonstrate improved activity in the aldol reaction between cyclohexanone and 4-nitrobenzaldehyde through a combination of rational methods.\nWhile the strategy is conceptually interesting, I do not find the novelty and impact of the approach sufficiently strong to warrant publication in Nature Communications. Similar strategies using natural L-proline or secondary amine-based catalysis in (artificial) enzymes for iminium/enamine catalysis (and in particular for asymmetric aldol reactions) is established\nThe LmrR scaffold has previously been preciously used in organocatalytic transformations, including via non-canonical amino acid incorporation or covalent modifications. While the authors acknowledge this, their argument that using natural residues is more desirable would benefit from further support. In particular, the scaffold already shows high conversion (albeit low enantioselectivity, ~7% ee), likely due to native lysine residues. The authors attribute this to three specific lysines but do not experimentally investigate their individual contributions. Creating alanine mutants at these positions would have strengthened the mechanistic understanding and might reveal catalytic potential of native residues that could be directly leveraged instead of introducing an complicated introduction of N-terminal proline. Moreover, the strategy of introduction of N-terminal L-proline appears to rely heavily on specific structural rearrangements to position the N-terminus correctly, which may limit its generalizability and find the authors claim that this approach is broadly applicable across other scaffolds to be overstated.\nIn terms of the manuscript itself, there are several aspects that could benefit from further clarification or refinement. I list some of the points below.\nMain Manuscript\n1. I would suggest avoiding the term “reprogramming” in the title (and elsewhere in the manuscript), as it is commonly associated with genetic code reprogramming which is not the focus of this study. A term such as “engineering” may be more appropriate and less misleading.\n2. Please consider using brackets to clearly denote transition states in Figure 1a. This could improve accuracy of the mechanistic depiction.\n3. In your Results and Discussion section, the statement “we hypothesized that its hydrophobic pocket might support latent catalytic activity by providing a favorable microenvironment for certain reactions” is somewhat redundant. LmrR has already been shown to be a versatile scaffold capable of providing such a microenvironment (as you also discuss in the Introduction). This is not a novel hypothesis you had.\n4. References 14 and 42 are the same.\n5. As mentioned earlier, I find it surprising that you did not prepare individual alanine mutants of the three lysine residues closest to the active site. This would be an important experiment to better understand their contribution to LmrR's activity and enantioselectivity.\n6. Your discussion attributes the low stereoselectivity to the achiral nature of lysine. However, this seems oversimplified. There are examples (for example recent work on DERA enzymes with active site lysine) where stereoselectivity is introduced by the surrounding chiral environment rather than the catalytic residue alone. A more nuanced discussion would be appreciated.\n7. You mention the use of directed evolution, but I would categorize all your experiments as rational design. Please reconsider your use of this term to avoid overstating your approach.\n8. There are several points where the narrative becomes confusing, particularly with the introduction of mutants. For instance, the LPEK4 variant appears multiple times before you explain its design and derivation. Consider restructuring for better logical flow.\n9. The catalytic efficiency of LPEK4 is significantly lower than the starting construct. While you introduce this, it is not discussed much. I encourage you to include a more thorough interpretation of this observation.\nSupporting Information (SI)\n1. You refer to your starting point as LmrR wild type, but this is not strictly correct. Your ‘’wild-type’’ version contains two mutations (K55D and K59Q) introduced in previous work by Roelfes. For actual wild-type LmrR please read for example here: https://doi.org/10.1038/emboj.2008.263. I suggest you clarify this point a bit better.\n2. Are all structural models prepared using AlphaFold? This is not clearly stated in the majority of your figures. I suggest including a paragraph in the Methods section explaining how structural models were obtained.\n3. The NMR spectra of your final compounds appear to show quite some impurities. Could you assign the peaks and provide an approximate estimate of the purity?\n4. Would it be possible to report enantiomeric excess (ee) values for LmrR catalysis in SI Figure 10? For some substrates, the ee appears quite significant so I think would be more accurate for comparison to include it."}, "author_response": "Point-by-point response to reviewer comments:\nComments in italics\nResponses in blue\nChanges in the body of the manuscript are noted in red.\nReviewer Comments\nReviewer #1 (Remarks to the Author):\nIn this manuscript, the authors report the redesign of the nonenzymatic protein scaffold\nLmrR into a non-natural enantioselective aldolase that uses an N-terminal Pro-1\nresidue as the key catalytic residue. The engineering of L-proline-based enzymes for\nasymmetric C-C bond formation (including enantioselective aldol reactions) has\nalready been achieved by Poelarends and coworkers with 4-oxalocrotonate\ntautomerase (4-OT). However, I do not think this diminishes the impact since 4-OT is\na truly exceptional protein, so achieving this with a different nonenzymatic protein is\nnot necessarily expected. Furthermore, the applied design strategy is interesting and\nunique. The authors engineered the LmrR sequence by removing the first four residues,\nexposing its native L-proline residue at the N-terminus, and subsequently deleting\nthree additional residues to shift the exposed N-terminal L-proline deeper into the\nhydrophobic pocket. This was followed by directed evolution and optimization of\nreaction conditions to improve performance. The authors explored the substrate scope\nusing cyclohexanone and several benzaldehyde derivatives, demonstrating that the\nenzyme can be used to produce a range of enantioenriched aldol products.\nThis work is interesting and original in terms of the applied enzyme design strategy,\nbut the following comments need to be addressed before publication:\nResponse: We thank you for your thoughtful and thorough evaluation of our manuscript.\nWe are glad that you found our work interesting and unique, particularly in terms of the\napplied enzyme design strategy. We greatly appreciate your constructive comments\nand have carefully considered and addressed each of them point-by-point below.\nThe authors mention that there findings reveal a unique strategy to create tailored, L-\nproline based enzymes for asymmetric synthesis. The authors should clearly mention\nthat their strategy appears to be limited to protein sequences with a proline residue\nclose to the N-terminus, which can also be relocated into a hydrophobic pocket.\nAlternatively, a Pro-1 residue can be directly introduced at the N-terminus, followed by\nrepositioning into a hydrophobic cavity.\nResponse: Thank you for this valuable suggestion. We agree that our design strategy\nis inherently influenced by the presence and position of proline residues in the protein\nscaffold. We also appreciate your insightful recommendation to highlight the alternative\nand more generalizable approach of introducing a proline residue directly at the N-\nterminus and repositioning it into a hydrophobic pocket.\nTo clarify this point, we have added the following text to the revised manuscript (Page\n3):\n“While demonstrated successfully with LmrR, this enzyme design strategy depends on\nthe presence and position of proline residues within the scaffold protein. In cases\nwhere a suitable proline is absent, a proline residue could be directly introduced at the\nN-terminus and subsequently repositioned into a hydrophobic cavity to achieve\ncatalysis and stereoselective chemical transformations.”\nThe authors have demonstrated that the three lysine residues proximal to the active\nsite primarily account for LmrR’s intrinsic aldolase activity. Why did the authors not use\nK6 as the key catalytic residue to evolve an artificial aldolase? This seems much\nsimpler than the applied strategy of relocating Pro-5 to the N-terminus, followed by\nplacing it deeper into LmrR’s hydrophobic pocket? The authors should discuss both\npotential strategies. Also, the argument that a proline residue (secondary amine) would\nbe better to achieve high stereoselectivity compared to a lysine residue (primary amine)\nperhaps makes sense for organocatalysis, but not necessarily for enzymatic reactions.\nNote that the chiral environment of the active site largely determines stereocontrol, as\nshown by the highly enantioselective aldol reactions catalyzed by many lysine-\nbased aldolases. This should be more carefully discussed as well.\nResponse: Thank you for this insightful and important comment. We agree that using\nK6 as the catalytic residue represents a plausible alternative to our Pro1-based design\nstrategy. During the revision, we constructed an LmrR variant (LmEK_A6K) in which\nonly K6 is retained in the hydrophobic pocket. This variant restored the intrinsic\naldolase activity (96% conversion), comparable to LmrR with all three lysines. However,\nits enantioselectivity remained low (11% e.e.). As also requested by Reviewer 2, we\nprepared additional variants retaining one or two lysines in the pocket. As shown in\nFigure R1, all such variants displayed consistently high conversions (93-96%) but\nuniformly poor enantioselectivity (6-11% e.e.).\nThese observations indicate that (i) any of the three lysines (K6, K22, and K101) can\nfunction as catalytic residues for the aldol reaction, but (ii) the LmrR pocket in its\ncurrent form does not provide an appropriate chiral environment for stereocontrol. We\nagree that many natural lysine-based aldolases achieve excellent stereoselectivity\nthrough a highly preorganized chiral active site. Our data suggest that achieving\ncomparable stereocontrol with lysine in LmrR would require substantial structural\nredesign of the binding pocket.\nOur intention was not to argue that proline is inherently superior to lysine in enzyme\ncatalysis. Rather, our goal was to develop and demonstrate a distinct, previously\nunexplored design concept: embedding an N-terminal proline-based enamine catalytic\nmotif within a tunable protein scaffold. This strategy provides a complementary route\nto stereocontrol that does not rely on the extensive catalytic networks present in natural\nlysine-dependent aldolases.\nTo address this comment, we have added the following text to the revised manuscript\n(Page 5):\n“We next evaluated LmrR variants retaining one or two lysine residues in the\nhydrophobic pocket. All such variants showed high conversions (93-96%) but low\nenantioselectivities (6-11% e.e.) (Supplementary Fig. 1). These findings indicate that\nalthough lysine residues (K6, K22, K101) can serve as catalytic nucleophiles, the LmrR\npocket does not provide the chiral environment necessary for effective stereocontrol.\nWhile natural lysine-dependent aldolases achieve high enantioselectivity through\nhighly organized active sites48-50, engineering LmrR to create a similarly preorganized\nchiral pocket around lysine would require extensive structural redesign. Therefore, in\nthis work we pursued an alternative and complementary strategy, leveraging proline-\nmediated enamine catalysis to introduce a chiral catalytic element directly into the\nactive site.”\nBelow is Figure R1 (these new data are now included in Supplementary Fig. 1):\na\nLmrR_K6A LmrR_K22A LmrR_K101A\n+2 mod. M M c o a b l s c 1 1 4 4 6 6 9 9 6 6 . . 1 2 1 6 6 7 +2 mod. M M c o a b l s c 1 1 4 4 6 6 9 9 5 6 . . 0 2 4 6 0 7 +2 mod. M M c o a b l s c 1 1 4 4 6 6 9 9 7 6 . . 8 2 7 6 5 7\nLmrR_K6A_mod. LmrR_K22A_mod. LmrR_K101A_mod.\nM M c o a b l s c 1 1 4 4 8 8 6 6 0 0 . . 1 4 8 2 3 3 M M c o a b l s c 1 1 4 4 8 8 5 6 9 0 . . 2 4 1 2 7 3 M M c o a b l s c 1 1 4 4 8 8 6 6 3 0 . . 4 4 6 2 7 3\n12500 15000 17500 12500 15000 17500 12500 15000 17500\nm/z m/z m/z\nLmEK_A6K LmEK_A22K LmEK_A101K\n+1 mod. Mcalc 14639.173 Mcalc 14639.173 Mcalc 14639.173\nMobs 14639.187 +1 mod. Mobs 14638.461 +1 mod. Mobs 14639.162\nLmEK_A6K_mod. LmEK_A22K_mod. LmEK_A101K_mod.\nMcalc 14721.251 Mcalc 14721.251 Mcalc 14721.251\nMobs 14720.329 Mobs 14720.138 Mobs 14720.530\n12500 15000 17500 12500 15000 17500 12500 15000 17500\nm/z m/z m/z\nb\n100 100\n80 80\n)%\n(\nn 60 60 )%\no\nis\nre\nv 40 40\n.e\n.\n(\ne\nn\no\nC\n20 20\n0 0\nL mr R L mr R_ K6 A L mr R_ K22 A L mr R_ K101 A L m E K L m E K_ A6 K L m E K_ A22 L K m E K_ A101 K\nFigure R1. Catalytic activity of LmrR variants retaining one or two lysine residues in the\nhydrophobic pocket. a, MALDI-TOF-MS analysis of LmrR variants before and after enamine\ntrapping. b, Substrate conversion and enantioselectivity of aldol products catalyzed by the\ndifferent variants. Data are shown as means, with error bars representing the standard\ndeviation from three independent experiments (n = 3).\nThe authors claim that in the engineered LPEK1–LPEK4 enzymes, the N-terminal L-\nproline functions as the sole ‘organocatalyst’ mediating enamine-based catalysis,\nindicating that the secondary amine of the Pro-1 residue is crucial for the improved\nperformance. In the best variant (LPEK4), the authors should make the Pro-1 to Ala-1\nmutation to demonstrate that indeed the secondary amine of Pro-1 is required rather\nthan an N-terminal primary amine (as present in Ala-1). Would a primary amine group\nwork equilly well? This is an important point to experimentally address because the\nauthors claim a unique engineering strategy to develop L-proline-based enzymes, with\na catalytically competent secondary amine, for asymmetric transformations.\nResponse: Thank you for raising this important point. To directly test whether the N-\nterminal secondary amine of Pro-1 is required for stereocontrol, we generated the\nvariant LPEK4_P1A, in which Pro-1 was replaced with Ala-1. As shown in Figure R2,\nLPEK4_P1A retained high catalytic activity (92% conversion), consistent with catalysis\nmediated by the N-terminal primary amine of alanine. However, its enantioselectivity\nremained low (11% e.e.), comparable to the lysine-dependent variant LmEK_A6K (96%\nconversion, 11% e.e.; Figure R1).\nThese results demonstrate that while a primary amine at the N-terminus can support\ncatalysis (and thus turnover), it cannot provide the stereocontrol achieved with the N-\nterminal secondary amine of proline (LPEK4: 73% e.e.). This experimentally confirms\nthat the improved enantioselectivity of LPEK variants relies on the unique catalytic\ngeometry provided by Pro-1.\nTo clarify this key point, we have added the following text to the revised manuscript\n(Page 8):\n“To examine whether an N-terminal primary amine could also mediate stereoselective\ncatalysis, we constructed LPEK4_P1A, in which the N-terminal proline was replaced\nwith alanine. Although LPEK4_P1A retained high catalytic activity (92% conversion),\nits enantioselectivity remained low (11% e.e.) (Supplementary Fig. 6). These results\nindicate that the N-terminal secondary amine of proline is essential for stereocontrol in\nthe LPEK variants.”\nBelow is Figure R2 (these new data are now included in Supplementary Fig. 6):\na b\n100 100\nLPEK4_P1A\nM 13708.102\ncalc\n+1 mod. M obs 13707.665 ) % 80 80\nM ca l L c P 1 E 37 K 9 4 0 _ . P 18 1 0 A_mod. ( n o 60 60 ) %\nM\nobs\n13788.174 is\nr\ne v 40 40\n.e\n.\n(\ne\nn\no\nC\n20 20\n0 0\n12500 15000 17500 LPEK4 LPEK4_P1A\nm/z\nFigure R2. Catalytic evaluation of LPEK4_P1A. a, MALDI-TOF-MS analysis of LPEK4_P1A\nbefore and after enamine trapping. b, Substrate conversion and enantioselectivity of aldol products catalyzed by LPEK4_P1A, with LPEK4 included as a control. Data are shown as\nmeans, with error bars representing the standard deviation from three independent experiments\n(n = 3).\nTo optimize the LPEK4-catalyzed aldol reactions, optimal reaction conditions were\ndetermined. How does the selected pH of 5.8 relate to the pKa value of the Pro-1\nresidue? It would be informative to determine the pKa of the pro-1 residue. The authors\nshould also make clear in the mansuscript that the placement of the Pro-1 residue in\na hydrophobic pocket is crucial to lower its pKa value.\nResponse: Thank you for this insightful comment. To address it, we determined the\npK value of the Pro-1 residue using both computational prediction and experimental\na\nmeasurement.\nFirst, pK values were predicted using the H++ server (http://biophysics.cs.vt.edu/H++).\na\nPro-1 in LPEK1 and LPEK4 showed predicted pK values of 7.0 and 5.5, respectively\na\n(Figure R3), indicating that repositioning Pro-1 deeper into the hydrophobic pocket\nsubstantially lowers its pK . This shift is consistent with the expected destabilization of\na\nthe protonated amine in a nonpolar microenvironment.\nSecond, using established experimental approaches (Giger et al., Nat. Chem. Biol.\n2013; Obexer et al., Nat. Chem. 2017), we determined the pH-rate profile of LPEK4-\ncatalyzed reactions. The experimentally derived pK of Pro-1 was 5.6 (Figure R4), in\na\nexcellent agreement with the predicted value of 5.5. At the reaction pH of 5.8, Pro-1 is\ntherefore optimally poised for deprotonation, enabling efficient enamine formation and\ncatalysis.\nTogether, these analyses demonstrate that placement of Pro-1 within the hydrophobic\npocket is crucial for lowering its pK to a catalytically competent range.\na\nGiger, L. et al. Evolution of a designed retro-aldolase leads to complete active site remodeling. Nat. Chem.\nBiol. 9, 494-498 (2013)\nObexer, R. et al. Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase. Nat.\nChem. 9, 50-56 (2017).\nTo make this clear, the following text has been added to the revised manuscript (Page\n10):\n“In addition, the pK of the P1 residue in LPEK4 was experimentally determined to be\na\n5.6 (Supplementary Fig. 13), consistent with its predicted value (pK 5.5) obtained\na\nusing H++62,63. At the reaction pH (5.8), P1 is therefore predominantly in the\ndeprotonated form, facilitating enamine formation. By comparison, P1 in LPEK1 has a\nhigher predicted pK (7.0), indicating that repositioning P1 into the hydrophobic pocket\na\nof LPEK4 is essential for lowering its pK and achieving effective catalysis.”\na\nBelow is Figure R3 (as noted for the reviewer, Figure R3 is provided only for evaluation\nand is not included in the manuscript; the pK values themselves are incorporated into\na\nthe revised text as described above):\nLPEK1 LPEK4\nFigure R3. Prediction of pK values of the Pro-1 residue in LPEK1 (left) and LPEK4 (right).\na\npK predictions were obtained using the H++ web server (http://biophysics.cs.vt.edu/H++). “NT”\na\ndenotes the N-terminal residue (Pro-1). The predicted pK values for Pro-1 are 7.0 in LPEK1\na\nand 5.5 in LPEK4, indicating that relocation of Pro-1 into the hydrophobic pocket substantially\nlowers its pK .\na\nBelow is Figure R4 (these new data are now included in Supplementary Fig. 13):\na b c\n15×10−3 20×10−3 20×10−3\npH 3.0 pH 5.0 pH 5.8\n15\n10 15\n10\n1-s\nE\n(\n[ v\n)\n/ ] 0 t −5 0\n5\nk K c m at = = 8 1 . . 2 4 m × M 10-2 s-1\n1-s\nE\n(\n[ v\n)\n/ ] 0 t − − 10 5 0\n5\nk K c m at = = 8 1 . . 0 7 m × M 10-2 s-1\n1-s\nE\n(\n[ v\n)\n/ ] 0 t\n10\n5 k K c m at = = 2 1 . . 2 9 m × M 10-2 s-1 MEPVKRRA m d ql me- o o Sj u d d t . a q a u e x Ru t c li - o ae S n rd qe u C(aCh r iOe -SDq)r y = 02 M .. V 02 imc 13 v\\ h 83 a- 100 a 52 (x0 ... e 78 588 )*l / 284 i ±± s [x 234 E M 00657 /\\ .. - e 431 ( 07 (Kn t07 ) tm]19 e 10 n + 86 x)\n0\n−10 −15\n0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35\n4-nitrobenzaldehyde (mM) 4-nitrobenzaldehyde (mM) 4-nitrobenzaldehyde (mM)\nd e f\n25×10−3 30×10−3\n16\npH 6.6 pH 7.3\n1-s\nE\n(\n[ v\n)\n/ ] 0 t1\n1 2\n0\n5 0\n5 k K c m at = = 1 1 . . 8 9 m × M 10-2 s-1\n1-s\nE\n(\n[ v\n)\n/ ] 0 t 1\n1 2 2\n0\n5 0 5\n5 k K c m at = = 2 2 . . 8 4 m × M 10-2 s-1\n1 1 - - M s\nK ( k\n)\n/ ta m c\n1 1 1\n2 4 6\n8 0 2 4 ( p k K ca a t / = K m 5 ) .6 max = 10.8 M-1 s-1\n0 0 0\n0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 2 3 4 5 6 7 8\n4-nitrobenzaldehyde (mM) 4-nitrobenzaldehyde (mM) pH\nFigure R4. Experimental determination of the pK of the Pro-1 residue in LPEK4. a-e,\na\nMichaelis-Menten plots for the LPEK4-catalyzed aldol reaction using 4-nitrobenzaldehyde as\nthe substrate at different pH values. f, pH-rate profile of the reaction, fitted to the equation:\n(k /K ) = (k /K ) /(1 + 10pKa–pH). The resulting fit yields the experimental pK of the Pro-\ncat m obs cat m max a\n1 residue in LPEK4. Data are shown as means, with error bars representing the standard\ndeviation from three independent experiments (n = 3).\nThroughout the manuscript the authors mention that the artificial aldolase catalyzes an\naldol condensation reaction. I believe it is more appropriate to call it an aldol addition\nreaction, as no dehydration of the aldol product takes place.\nResponse: Thank you for this important clarification. We fully agree that the reactions\ndescribed in our manuscript should be referred to as aldol addition rather than aldol\ncondensation reactions, as no dehydration step occurs under our reaction conditions\nand the isolated products are β-hydroxy ketones rather than α,β-unsaturated ketones.\nIn accordance with your suggestion, we have revised the terminology throughout the\nmanuscript, replacing “aldol condensation” with “aldol addition” where appropriate.\nP2/3: “One of the few known exceptions is 4-oxalocrotonate tautomerase (4-OT),\nwhich possesses an N-terminal L-proline that mediates secondary amine catalysis and\nhas been engineered for asymmetric reactions38-41.” Here the authors should be\nmore accurate and include the following references on 4-OT catalyzed asymmetric\ntransformations (Angew Chem Int Ed Engl. 2022 Feb 14;61(8):e202113970. doi:\n10.1002/anie.202113970; Angew Chem Int Ed Engl. 2021 Nov 2;60(45):24059-24063;\nAngew Chem Int Ed Engl. 2020 Jun 22;59(26):10374-10378. doi:\n10.1002/anie.202001373).\nResponse: Thank you for pointing this out and highlighting these important additional\nreferences. We agree that the statement should be more precise and should reflect\nthe full scope of asymmetric transformations catalyzed by engineered 4-OT variants.\nAccordingly, we have incorporated the suggested citations into the revised manuscript\n(Pages 2-3):\n“One of the few known exceptions is 4-oxalocrotonate tautomerase (4-OT), which\npossesses an N-terminal L-proline that mediates secondary amine catalysis and has\nbeen engineered for asymmetric reactions38-44.”\n42. Xu, G., Crotti, M., Saravanan, T., Kataja, K. M. & Poelarends G. J.\nEnantiocomplementary epoxidation reactions catalyzed by an engineered cofactor-\nindependent non-natural peroxygenase. Angew. Chem. Int. Ed. 59, 10374-10378\n(2020).\n43. Kunzendorf, A., Xu, G., Saifuddin, M., Saravanan, T. & Poelarends, G. J.\nBiocatalytic asymmetric cyclopropanations via enzyme-bound iminium ion\nintermediates. Angew. Chem. Int. Ed. 60, 24059-24063 (2021).\n44. Xu, G. et al. Gene fusion and directed evolution to break structural symmetry and\nboost catalysis by an oligomeric C-C bond-forming enzyme. Angew. Chem. Int. Ed. 61,\ne202113970 (2022).\nP11: “Given the broad utility of L-proline-based organocatalysis in asymmetric\nsynthesis….” Here the authors should also mention that there is a large body of\nliterature showing the broad utility of L-proline-based biocatalysts in asymmetric\nsynthesis (work on 4-OT by Poelarends and coworkers).\nResponse: Thank you for this valuable suggestion. We have revised the sentence to\nclearly acknowledge the extensive work on L-proline-based biocatalysts, particularly\n4-OT developed by Poelarends and coworkers. The updated text now reads (Page 13):\n“Given the broad utility of L-proline-based organocatalysis28-37 and the demonstrated\neffectiveness of natural L-proline-based biocatalysts—most notably 4-oxalocrotonate\ntautomerase38-44 in asymmetric synthesis, ...”\nFrom the SI it is not clear how racemic and enantiopure reference compounds have\nbeen prepared? This seemingly missing information should be added to the SI. It is\nalso not clear how the absolute configuration has been determined? Spectra with\nappropriate references seem to be missing and should be added for at least a few\nproducts.\nResponse: Thank you for pointing out these important omissions. We have now added\nthe missing information to the Supplementary Information to ensure full clarity and\nreproducibility, as detailed below.\n(1) In the “Preparation and purification of chemicals” section, we have added the\nfollowing text:\n“Subsequently, the crude reaction mixtures containing racemic aldol products were\nseparated by preparative HPLC (Agilent Pursuit XRs 5 C18, 250 × 10 mm), yielding\ntwo diastereomeric fractions (anti and syn). Each diastereomer was characterized by\nNMR spectroscopy to confirm its configuration. Enantiomers within each\ndiastereomeric pair were then resolved and analyzed by chiral HPLC (Chiralpak-AS-H\nor Chiralpak-IBN-5, 250 × 4.6 mm). These enantiopure chromatograms serve as\nreference standards for assigning absolute configuration of aldol products formed by\nLmrR variants.”\n(2) For each compound shown in the “Preparation and purification of chemicals”\nsection, we have added preparative HPLC chromatograms demonstrating separation\nof the anti and syn diastereomers, as well as complete 1H NMR data for structural\nconfirmation.\n(3) In the “Characterization and analytical methods” section, we have added methods\nfor NRM analysis.\n“Nuclear magnetic resonance (NMR) spectroscopy\nNMR spectra were acquired on a Bruker Avance NEO 400 MHz spectrometer.\nSamples were dissolved in CDCl and chemical shifts (ppm) were referenced to\n3\ntetramethylsilane (TMS, internal standard). Data were processed using MestReNova\nx64 (version 14.3.1).”\n(4) For your convenience, we provided below a representative workflow (Figure R5)\nfor compound 3a (anti and syn). Please note that this figure is presented here solely\nfor your reference. All corresponding HPLC chromatograms, NMR spectra, and other\ndetails have been included in the Supplementary Information where appropriate.\na b\n3000 anti-3a syn-3a\nanti-3a\n2500\n2000\n1500\n1000\n500\n0\n30 31 32 33 34 35 36 37 38 39 40\nTime (min)\nc\n3.3 anti-3a\n2.2\n1.1\n0.0\nsyn-3a\nsyn-3a\n16.8\n11.2\n5.6\n0.0\nLmrR\n20.7\n13.8\n6.9\n0.0\nLPEK4\n20.7\n13.8\n6.9\n0.0\n25 30 35 40 45 50 55 60\nTime (min)\nFigure R5. Characterization of aldol products generated by LmrR and its variants. a,\nPreparative HPLC chromatograms showing separation of the anti and syn diastereomers of\nproduct 3a. b, 1H NMR spectra of isolated diastereomers anti-3a and syn-3a, confirming their\nstructural assignments. c, Chiral HPLC analyses of enantiomers (top to bottom: enantiopure\nanti-3a, enantiopure syn-3a, product mixture from LmrR, and product mixture from LPEK4),\nused to determine the enantiomeric composition and absolute configuration of enzymatically\ngenerated products.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Lu et al. presents an interesting example of engineering the LmrR\nprotein scaffold to feature an N-terminal L-proline residue capable of participating in\nenamine-based aldol catalysis. The central innovation lies in repositioning the N-\nterminus so that a canonical L-proline becomes catalytically exposed, eliminating the\nneed for non-canonical amino acids or post-translational modifications to introduce a\nsecondary amine. The authors further demonstrate improved activity in the aldol\nreaction between cyclohexanone and 4-nitrobenzaldehyde through a combination of\nrational methods.\nResponse: We sincerely thank you for the thorough and thoughtful evaluation of our\nmanuscript, and for highlighting both the interest and innovation of our study. We are\ngrateful for your constructive comments, which have greatly helped us improve the\nclarity and rigor of the work. We address all points in detail below.\nWhile the strategy is conceptually interesting, I do not find the novelty and impact of\nthe approach sufficiently strong to warrant publication in Nature Communications.\nSimilar strategies using natural L-proline or secondary amine-based catalysis in\n(artificial) enzymes for iminium/enamine catalysis (and in particular for asymmetric\naldol reactions) is established The LmrR scaffold has previously been preciously used\nin organocatalytic transformations, including via non-canonical amino acid\nincorporation or covalent modifications. While the authors acknowledge this, their\nargument that using natural residues is more desirable would benefit from further\nsupport. In particular, the scaffold already shows high conversion (albeit low\nenantioselectivity, ~7% ee), likely due to native lysine residues. The authors attribute\nthis to three specific lysines but do not experimentally investigate their individual\ncontributions. Creating alanine mutants at these positions would have strengthened\nthe mechanistic understanding and might reveal catalytic potential of native residues\nthat could be directly leveraged instead of introducing an complicated introduction of\nN-terminal proline. Moreover, the strategy of introduction of N-terminal L-proline\nappears to rely heavily on specific structural rearrangements to position the N-terminus\ncorrectly, which may limit its generalizability and find the authors claim that this\napproach is broadly applicable across other scaffolds to be overstated.\nResponse: Thank you very much for your careful evaluation and constructive feedback.\nWe appreciate the opportunity for clarification and address the comments below.\nFirst, we fully agree that iminium/enamine catalysis in designed enzymes—particularly\nvia non-canonical amino acids or covalent cofactor installation—has been successfully\ndemonstrated. Our objective here was not to replicate these strategies, but rather to\ndevelop a fundamentally different design principle: creating a catalytically competent\nsecondary amine solely through protein backbone engineering, without the use of non-\ncanonical amino acids, chemical installation, or external cofactors.\nTo our knowledge, repositioning an endogenous proline residue to the N-terminus and\nrelocating it into a hydrophobic pocket via sequence truncation and rearrangement\nalone has not been previously reported. This strategy enables the generation of an\nintrinsic, genetically encoded organocatalyst and eliminates reliance on non-natural\ncomponents—an important advantage when considering evolvability, scalability, and\ncompatibility with cellular expression systems.\nSecond, we appreciate your suggestion to experimentally dissect the contributions of\nthe three lysine residues (K6, K22, and K101). During revision, we constructed LmrR\nvariants retaining one or two lysine residues in the hydrophobic pocket and evaluated\ntheir catalytic performance. All variants showed similarly high conversions (93-96%)\nbut uniformly low enantioselectivities (6-11% e.e.), regardless of which lysine(s) were\npresent. These results demonstrate that while lysines can catalyze aldol addition, the\nLmrR pocket lacks the preorganized chiral environment required for stereocontrol—\nunlike natural lysine-dependent aldolases. Although stereocontrol around lysine could\nin principle be engineered, it would require extensive active-site redesign. Instead, we\npursued a complementary strategy by introducing a chiral catalytic element (N-terminal\nproline) to directly enhance stereoselectivity. These new data and analyses have been\nadded to the revised manuscript (Supplementary Fig. 1; Page 5). For more details,\nplease see below our response to Comment 5.\nFinally, we agree that the applicability of this strategy is scaffold-dependent and have\nrevised the manuscript to avoid overgeneralization. Our intent is not to claim universal\napplicability, but rather to demonstrate a rational pathway for repurposing endogenous\nresidues—particularly proline—for asymmetric catalysis. We now clearly state that this\napproach is most suitable for scaffolds with engineerable N-termini near hydrophobic\ncavities and that, where necessary, proline can be introduced at the N-terminus and\nrepositioned through modest truncation or loop engineering.\nTo clarify these points, we have strengthened the discussion in the revised manuscript\nand removed the text implying broad universal applicability as follows:\n(1) Text added (Page 3): “While demonstrated successfully with LmrR, this enzyme\ndesign strategy depends on the presence and position of proline residues within the\nscaffold protein. In cases where a suitable proline is absent, a proline residue could be\ndirectly introduced at the N-terminus and subsequently repositioned into a hydrophobic\ncavity to achieve catalysis and stereoselective chemical transformations.”\n(2) Text added (Page 12): “However, this enzyme design strategy is likely limited to\nprotein scaffolds containing a proline residue near the N-terminus that can be\nrepositioned into a hydrophobic pocket. Nevertheless, it enables the creation of an\nintrinsic, genetically encoded organocatalyst without reliance on nonnatural\ncomponents, offering distinct advantages in evolvability, scalability, and compatibility\nwith cellular expression systems.”\n(3) Text deleted: “More broadly, it establishes a new approach for leveraging native\nresidues such as proline in protein scaffolds to develop artificial enzymes for\nstereoselective chemical transformations.”\n(4) Text modified (Pages 12-13): “Altogether, our study demonstrates the potential of\nnatural protein scaffolds such as LmrR with engineerable termini adjacent to a\nhydrophobic cavity for building robust, tunable, and enantioselective biocatalysts.”\nWe are grateful for your insightful feedback, which has significantly improved the clarity,\nmechanistic understanding, and positioning of our work.\nIn terms of the manuscript itself, there are several aspects that could benefit from\nfurther clarification or refinement. I list some of the points below.\nMain Manuscript\n1. I would suggest avoiding the term “reprogramming” in the title (and elsewhere in the\nmanuscript), as it is commonly associated with genetic code reprogramming which is\nnot the focus of this study. A term such as “engineering” may be more appropriate and\nless misleading.\nResponse: Thank you for this helpful suggestion. We agree that “reprogramming” may\nbe misleading in this context. Accordingly, we have replaced “reprogramming” with\n“engineering” in the title and throughout the manuscript to improve clarity and avoid\npotential confusion.\n2. Please consider using brackets to clearly denote transition states in Figure 1a. This\ncould improve accuracy of the mechanistic depiction.\nResponse: Thank you for this important comment. To improve the accuracy and clarity\nof the mechanistic depiction, we have added brackets to denote transition states in\nFigure 1a (see below). Please refer to the updated Figure 1 in the revised manuscript.\na\n3. In your Results and Discussion section, the statement “we hypothesized that its\nhydrophobic pocket might support latent catalytic activity by providing a favorable\nmicroenvironment for certain reactions” is somewhat redundant. LmrR has already\nbeen shown to be a versatile scaffold capable of providing such a microenvironment\n(as you also discuss in the Introduction). This is not a novel hypothesis you had.\nResponse: Thank you for this thoughtful comment. We agree that this statement could\nbe interpreted as redundant, given prior work establishing LmrR as a versatile catalytic\nscaffold. To avoid any misunderstanding and to better align with the existing literature,\nwe have revised the wording by removing “we hypothesized that” and rephrasing the\nsentence as follows (Page 4):\n“Although LmrR lacks any known native enzymatic function14,19,25, its hydrophobic\npocket alone may nonetheless support latent catalytic activity.”\n4. References 14 and 42 are the same.\nResponse: Thank you for pointing this out. We have removed ref. 42 and updated the\nreference list.\n5. As mentioned earlier, I find it surprising that you did not prepare individual alanine\nmutants of the three lysine residues closest to the active site. This would be an\nimportant experiment to better understand their contribution to LmrR's activity and\nenantioselectivity.\nResponse: We appreciate this insightful and constructive comment. During revision,\nwe performed additional experiments to systematically evaluate the contributions of\nthe three lysine residues proximal to the active site (K6, K22, and K101).\nSpecifically, we constructed a series of LmrR variants in which one or two lysine\nresidues were retained in the hydrophobic pocket and assessed their catalytic\nperformance in the aldol reaction. As shown in Figure R6, all variants exhibited\nsimilarly high conversions (93-96%), yet uniformly low enantioselectivities (6-11% e.e.),\nindependent of which lysine residue(s) were present. These results indicate that (i)\neach of the three lysines (K6, K22, and K101) can serve as a catalytic nucleophile for\naldol addition, but (ii) the native LmrR pocket does not provide a sufficiently organized\nchiral environment to impart effective stereocontrol.\nAs discussed above, while it is in principle possible to engineer stereocontrol around\nlysine residues—as achieved in natural lysine-dependent aldolases—doing so in LmrR\nwould likely require extensive redesign of the binding pocket to establish the necessary\npreorganized chiral architecture. Instead, in this study we intentionally pursued a\ncomplementary design strategy by introducing a chiral catalytic element (an N-terminal\nproline) directly into the hydrophobic pocket to enhance stereoselectivity without large-\nscale structural remodeling.\nTo address this comment, we have added the following text to the revised manuscript\n(Page 5):\n“We next evaluated LmrR variants retaining one or two lysine residues in the\nhydrophobic pocket. All such variants showed high conversions (93-96%) but low\nenantioselectivities (6-11% e.e.) (Supplementary Fig. 1). These findings indicate that\nalthough lysine residues (K6, K22, K101) can serve as catalytic nucleophiles, the LmrR\npocket does not provide the chiral environment necessary for effective stereocontrol.\nWhile natural lysine-dependent aldolases achieve high enantioselectivity through\nhighly organized active sites48-50, engineering LmrR to create a similarly preorganized\nchiral pocket around lysine would require extensive structural redesign. Therefore, in\nthis work we pursued an alternative and complementary strategy, leveraging proline-\nmediated enamine catalysis to introduce a chiral catalytic element directly into the\nactive site.”\nBelow is Figure R6 (these new data are now included in Supplementary Fig. 1):\na\nLmrR_K6A LmrR_K22A LmrR_K101A\n+2 mod. M M c o a b l s c 1 1 4 4 6 6 9 9 6 6 . . 1 2 1 6 6 7 +2 mod. M M c o a b l s c 1 1 4 4 6 6 9 9 5 6 . . 0 2 4 6 0 7 +2 mod. M M c o a b l s c 1 1 4 4 6 6 9 9 7 6 . . 8 2 7 6 5 7\nLmrR_K6A_mod. LmrR_K22A_mod. LmrR_K101A_mod.\nM M c o a b l s c 1 1 4 4 8 8 6 6 0 0 . . 1 4 8 2 3 3 M M c o a b l s c 1 1 4 4 8 8 5 6 9 0 . . 2 4 1 2 7 3 M M c o a b l s c 1 1 4 4 8 8 6 6 3 0 . . 4 4 6 2 7 3\n12500 15000 17500 12500 15000 17500 12500 15000 17500\nm/z m/z m/z\nLmEK_A6K LmEK_A22K LmEK_A101K\n+1 mod. Mcalc 14639.173 Mcalc 14639.173 Mcalc 14639.173\nMobs 14639.187 +1 mod. Mobs 14638.461 +1 mod. Mobs 14639.162\nLmEK_A6K_mod. LmEK_A22K_mod. LmEK_A101K_mod.\nMcalc 14721.251 Mcalc 14721.251 Mcalc 14721.251\nMobs 14720.329 Mobs 14720.138 Mobs 14720.530\n12500 15000 17500 12500 15000 17500 12500 15000 17500\nm/z m/z m/z\nb\n100 100\n80 80\n)%\n(\nn 60 60 )%\no\nis\nre\nv 40 40\n.e\n.\n(\ne\nn\no\nC\n20 20\n0 0\nL mr R L mr R_ K6 A L mr R_ K22 A L mr R_ K101 A L m E K L m E K_ A6 K L m E K_ A22 L K m E K_ A101 K\nFigure R6. Catalytic activity of LmrR variants retaining one or two lysine residues in the\nhydrophobic pocket. a, MALDI-TOF-MS analysis of LmrR variants before and after enamine\ntrapping. b, Substrate conversion and enantioselectivity of aldol products catalyzed by the\ndifferent variants. Data are shown as means, with error bars representing the standard\ndeviation from three independent experiments (n = 3).\n6. Your discussion attributes the low stereoselectivity to the achiral nature of lysine.\nHowever, this seems oversimplified. There are examples (for example recent work on\nDERA enzymes with active site lysine) where stereoselectivity is introduced by the\nsurrounding chiral environment rather than the catalytic residue alone. A more\nnuanced discussion would be appreciated.\nResponse: Thank you for this insightful comment. We fully agree that stereoselectivity\nin enzymatic aldol reactions is not dictated solely by the nature of the catalytic residue\nitself, but rather emerges from the interplay between the catalytic nucleophile and the\nsurrounding chiral environment of the active site. As you correctly note, several natural\nlysine-dependent aldolases, including DERA enzymes (Kunzendorf et al., ACS Catal.\n2021; Zhou et al. Angew. Chem. Int. Ed. 2025 – now cited as Refs. 48 and 49 in the\nrevised manuscript), achieve excellent stereocontrol through highly organized and\npreorganized active-site architectures.\nAs discussed in our response to Comment 5, we performed additional experiments to\nsystematically analyze the contributions of lysine residues within the LmrR scaffold.\nSpecifically, we constructed LmrR variants retaining one, two, or three lysine residues\nin the hydrophobic pocket. As shown in Figure R6 (see above), all variants exhibited\nsimilarly high catalytic activity but consistently low enantioselectivity. These findings\nindicate that, unlike natural lysine-based aldolases, the LmrR pocket lacks the precise\nspatial organization required to translate lysine-mediated catalysis into effective\nstereochemical control.\nAccordingly, we have revised the discussion to provide a more nuanced interpretation\nand to acknowledge the role of the protein environment in lysine-based aldolases. The\nrevised text now reads (Page 5):\n“We next evaluated LmrR variants retaining one or two lysine residues in the\nhydrophobic pocket. All such variants showed high conversions (93-96%) but low\nenantioselectivities (6-11% e.e.) (Supplementary Fig. 1). These findings indicate that\nalthough lysine residues (K6, K22, K101) can serve as catalytic nucleophiles, the LmrR\npocket does not provide the chiral environment necessary for effective stereocontrol.\nWhile natural lysine-dependent aldolases achieve high enantioselectivity through\nhighly organized active sites48-50, engineering LmrR to create a similarly preorganized\nchiral pocket around lysine would require extensive structural redesign. Therefore, in\nthis work we pursued an alternative and complementary strategy, leveraging proline-\nmediated enamine catalysis to introduce a chiral catalytic element directly into the\nactive site.”\n7. You mention the use of directed evolution, but I would categorize all your\nexperiments as rational design. Please reconsider your use of this term to avoid\noverstating your approach.\nResponse: Thank you for this important clarification. We agree that our approach is\nbest described as rational design/protein engineering rather than directed evolution.\nAccordingly, we have replaced the term “directed evolution” with “rational design” or\n“protein engineering” throughout the manuscript. In addition, consistent with this\nclarification, we have also revised related terminology (e.g., replacing “reprogramming”\nwith “engineering” in the title and main text) to avoid overstating the experimental\nstrategy.\n8. There are several points where the narrative becomes confusing, particularly with\nthe introduction of mutants. For instance, the LPEK4 variant appears multiple times\nbefore you explain its design and derivation. Consider restructuring for better logical\nflow.\nResponse: Thank you for this constructive comment and we appreciate your concern\nregarding narrative clarity, particularly in the introduction of engineered variants.\nIn our manuscript, Figure 1b,c is intentionally used to provide an early, schematic\noverview of the overall protein design strategy and engineering steps, allowing readers\nto grasp the conceptual framework before encountering detailed experimental results.\nThis figure summarizes the successive design steps that ultimately lead to the LPEK\nvariants.\nWe agree that mentioning specific variants before fully describing their design can be\nconfusing. In the section “Rational design of LmrR for high enantioselectivity”, the\nterm “LPEK1-LPEK4 enzymes” appeared prematurely in the final sentence of the second\nparagraph. To improve logical flow and avoid introducing LPEK4 before its derivation\nis explained, we have revised this wording to “LPEK variants” (Page 7) and ensured\nthat each variant is introduced after its design rationale and construction are clearly\ndescribed.\n9. The catalytic efficiency of LPEK4 is significantly lower than the starting construct.\nWhile you introduce this, it is not discussed much. I encourage you to include a more\nthorough interpretation of this observation.\nResponse: Thank you for this insightful comment. We agree that the reduced catalytic\nefficiency of LPEK4 relative to the starting construct merits further discussion, and we\nhave expanded our analysis accordingly.\nWe attribute the lower catalytic efficiency of LPEK4 primarily to the reduced number of\ncatalytically competent nucleophilic residues in the active site. Specifically, LPEK4\ncontains a single N-terminal proline as the catalytic residue, whereas LmrR possesses\nthree lysine residues (K6, K22, and K101) that can contribute to aldol catalysis. This\ninterpretation is supported by the observed kinetic parameters: the k of LPEK4 (2.5\ncat\nx 10-2 s-1) is about twofold lower than that of LmrR (4.4 x 10-2 s-1) (Supplementary Fig.\n11).\nTo further validate this point, and as described in our response to Comment 5, we\nconstructed additional LmrR variants (LmEK_A6K, LmEK_A22K, and LmEK_A101K),\neach retaining only a single lysine residue within the hydrophobic pocket. Kinetic\nanalyses of these variants revealed k values of 1.9-2.6 x 10-2 s-1 (see Figure R7\ncat\nbelow), comparable to that of LPEK4 and significantly lower than that of LmrR.\nMoreover, their K values increased to 2.8-4.1 mM, exceeding those of both LPEK4\nm\n(K = 2.2 mM) and LmrR (K = 1.7 mM). Consequently, the overall catalytic efficiencies\nm m\nof these single-lysine variants were also markedly reduced relative to LmrR.\nTogether, these findings indicate that the diminished catalytic efficiency of LPEK4 is\nnot solely attributable to the introduction of an N-terminal proline, but rather reflects a\ngeneral consequence of reducing the number of catalytically active residues within the\npocket. Importantly, this reduction in turnover is accompanied by a substantial gain in\nenantioselectivity, highlighting an inherent trade-off between catalytic efficiency and\nstereocontrol in this system.\nTo clarify this point, we have added the following text to the revised manuscript (Page\n8):\n“In addition, we measured the kinetic parameters of three LmrR variants (LmEK_A6K,\nLmEK_A22K, and LmEK_A101K), each retaining only a single lysine residue in the\nactive site (Supplementary Fig. 12). These variants also exhibited substantially\nreduced catalytic efficiencies (0.59-0.86 x 10-2 mM-1 s-1) compared to LmrR containing\nthree lysine residues. Together with the kinetic data for LPEK4, these observations\nsuggest that the reduced catalytic efficiency arises primarily from the reduced number\nof catalytically active residues in the pocket, rather than from the nature of the N-\nterminal proline itself. Yet importantly, this reduction in activity is accompanied by a\nsubstantial gain in enantioselectivity, highlighting the trade-off between turnover and\nstereochemical control.”\nBelow is Figure R7 (these new data are now included in Supplementary Fig. 12):\na b c\n30×10−3 30×10−3 25×10−3\nLmEK_A6K LmEK_A22K LmEK_A101K\n25 25 20\n1-s )20 1-s )20 1-s )15\nE\n(\n]t\n(\n]t\n(\n]t10\n[ v /0 10 5 k K c m at = = 4 2 . . 1 6 m × M 10-2 s-1 [ v /0 10 5 k K c m at = = 2 2 . . 8 4 m × M 10-2 s-1 [ v /0 5 k K c m at = = 3 1 . . 2 9 m × M 10-2 s-1\n0 0 0\n0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35\n4-nitrobenzaldehyde (mM) 4-nitrobenzaldehyde (mM) 4-nitrobenzaldehyde (mM)\nFigure R7. Kinetic characterization of (a) LmEK_A6K, (b) LmEK_A22K, and (c) LmEK_A101K.\nBased on the plot of v /[E] versus [4-nitrobenzaldehyde] at a cyclohexanone concentration of\n0 t\n200 mM, k and K values of LmEK_A6K, LmEK_A22K, and LmEK_A101K were calculated,\ncat m\nrespectively. Data are shown as means, with error bars representing the standard deviation\nfrom three independent experiments (n = 3).\nSupporting Information (SI)\n1. You refer to your starting point as LmrR wild type, but this is not strictly correct. Your\n‘’wild-type’’ version contains two mutations (K55D and K59Q) introduced in previous\nwork by Roelfes. For actual wild-type LmrR please read for example here:\nhttps://doi.org/10.1038/emboj.2008.263. I suggest you clarify this point a bit better.\nResponse: Thank you for raising this important point of clarification. We agree that the\nLmrR used in this study is not the authentic wild-type protein, as it contains the K55D\nand K59Q mutations introduced in earlier work by Roelfes and coworkers. To avoid\nany potential confusion, we have removed the term “wild-type” throughout the main\ntext and Supporting Information, and we now refer to this construct consistently as the\n“parental LmrR” where appropriate.\nIn the Supporting Information, the amino acid sequence, including these two mutations,\nis clearly provided, and the following clarifying text has been added: “parental protein\nderived from the wild-type protein, containing the K55D and K59Q mutations”.\n2. Are all structural models prepared using AlphaFold? This is not clearly stated in the\nmajority of your figures. I suggest including a paragraph in the Methods section\nexplaining how structural models were obtained.\nResponse: Thank you for this suggestion. All protein structural models presented in\nthis work were predicted using AlphaFold and subsequently visualized using PyMOL\nand, where applicable, additional web-based tools. To clearly document this, we have\nadded a dedicated paragraph to the supplementary Methods section as follows:\n“Prediction and visualization of protein structures\nAll protein structures were predicted using AlphaFold from their amino acid sequences\nand visualized with PyMOL (version 2.2.0), unless otherwise noted. Protein structural\ndynamics were evaluated using the CABS-flex web server\n(https://biocomp.chem.uw.edu.pl/CABSflex2). Molecular docking between enzymes\nand substrates was performed using the DockThor-VS web server\n(https://dockthor.lncc.br/v2/).”\nIn addition, we have updated the figure legends of Figs. 1-2 and Supplementary Figs.\n3-5 to state how the corresponding structural models were obtained.\n3. The NMR spectra of your final compounds appear to show quite some impurities.\nCould you assign the peaks and provide an approximate estimate of the purity?\nResponse: Thank you for this valuable and careful observation. We agree that the\nNMR spectra contained some impurity signals and that peak assignments were not\nsufficiently documented.\nUpon reanalysis, the major non-product signals can be attributed as follows: the peak\nat ~0.00 ppm corresponds to the internal standard tetramethylsilane (TMS); the signal\nat ~1.56 ppm arises from residual water; and the residual proton signal of deuterated\nchloroform (CDCl ) appears at ~7.26 ppm.\npresentation in Supplementary Information as follows:\n(1) We added a dedicated description of the NMR acquisition and analysis procedures:\n“Nuclear magnetic resonance (NMR) spectroscopy\nNMR spectra were acquired on a Bruker Avance NEO 400 MHz spectrometer.\nSamples were dissolved in CDCl and chemical shifts (ppm) were referenced to\n3\ntetramethylsilane (TMS, internal standard). Data were processed using MestReNova\nx64 (version 14.3.1).”\n(2) We added complete 1H NMR data to confirm the structures of all 24 enzymatically\nsynthesized and purified compounds (see the updated “Preparation and purification of\nchemicals” section for details).\n(3) We have added clarifying information on common impurity/reference peaks (TMS,\nresidual water, and CDCl₃) in the revised “NMR spectra” section as follows:\n“Note that, in the spectra below, the signal at ~0.00 ppm corresponds to the internal\nstandard tetramethylsilane (TMS), the signal at ~1.56 ppm arises from residual water,\nand the residual proton signal of CDCl appears at ~7.26 ppm.”\n3\n(4) All spectra in the revised Supplementary Information now include comprehensive\npeak assignments (see the updated “NMR spectra” section).\n4. Would it be possible to report enantiomeric excess (ee) values for LmrR catalysis in\nSI Figure 10? For some substrates, the ee appears quite significant so I think would\nbe more accurate for comparison to include it.\nResponse: Thank you for this valuable suggestion. We have included the enantiomeric\nexcess (e.e.) values in the updated figure as shown below (now it has been reordered\nas Supplementary Fig. 14).\nSupplementary Fig. 14 Substrate scope of LmrR-catalyzed enantioselective aldol\nreaction. Standard reaction conditions were as follows: substrates 1 (200 mM) and 2 (20 mM),\nLmrR enzyme (1.5 mol%), PBS buffer (10 mM, pH 7.3), reaction temperature 30 °C, and 12 h."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I would like to compliment the authors on their revision and am pleased with the changes made. I support publication of the revised manuscript without further experimentation and analysis."}, "author_response": ""}]

Decision letter