Engineering of Candida glabrata Ketoreductase 1 for Asymmetric

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Engineering of Candida glabrata Ketoreductase 1 for Asymmetric Reduction of #-Halo Ketones Fengyu Qin, Bin Qin, Takahiro Mori, Yan Wang, Lingxin Meng, Xin Zhang, Xian Jia, Ikuro Abe, and Song You ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b01552 • Publication Date (Web): 05 Aug 2016 Downloaded from http://pubs.acs.org on August 6, 2016

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Engineering of Candida glabrata Ketoreductase 1 for Asymmetric Reduction of α-Halo Ketones Fengyu Qin,†, ‡ Bin Qin, †, ‡ Takahiro Mori,¶,┴ Yan Wang,† Lingxin Meng,§ Xin Zhang,† Xian Jia,§ Ikuro Abe,¶ and Song You⃰ , † †

School of Life Sciences and Biopharmaceutical Sciences, Shenyang Pharmaceutical University,

103 Wenhua Road, Shenhe, Shenyang 110016, China ¶

Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-

ku, Tokyo 113-0033, Japan §

School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua

Road, Shenhe, Shenyang 110016, China

ABSTRACT Enantiopure halohydrins, which are important building blocks for pharmaceutical agents, could be synthesized by biocatalytic reduction of α-halo ketones using ketoreductases. In this study, Candida glabrata ketoreductase 1 (CgKR1) variants with >99% stereoselectivity toward α-halo ketones, such as 2-chloro-acetophenone, 2-chloro-4'-fluoroacetophenone and 2-bromoacetophenone, were obtained through engineering of CgKR1 at residues Phe92 and Tyr208. Interestingly, asymmetric reduction of these α-halo ketones by all the variants of CgKR1

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followed anti-Prelog’s rule, which is rarely found in natural ketoreductases. Moreover, the biocatalytic processes for reduction of these aromatic α-halo ketones with high substrate loading were achieved by co-expression of glucose dehydrogenase (GDH) for NADPH regeneration, indicating the potential of practical applications of these variants.

KEYWORDS: ketoreductases, protein engineering, asymmetric reduction, α-halo ketones, halohydrins

Introduction Ketoreductases are enzymes that catalyze the asymmetric reduction of prochiral carbonyl compounds to corresponding chiral alcohols in the presence of NAD(P)H.1-3 These enzymes usually display high stereoselectivity towards a wide range of substrates at mild reaction conditions.1-3 Due to their excellent selectivity and environment compatibility, ketoreductases are used commonly in industrial pharmaceutical synthesis in recent years.4,5 For instance, the biocatalytic processes with ketoreductases to synthesize the key intermediates for atorvastatin (Lipitor), montelukast (Singulair), crizotinib (Xalkori), duloxetine (Cymbalta), ezetimibe (Zetia) and atazanavir (Reyetaz) have been developed.4,5 Enantiopure halohydrins, which could be diversified into chiral diols, epoxides, amino alcohols and azido alcohols, are versatile building blocks for construction of a variety of pharmaceutical agents or agricultural chemicals. For instance, (R)-2-chloro-1-phenylethanol could be used for the synthesis of mirabegron (β-3 adrenergic receptor agonist),6 while (S)-2-chloro-1-(2,4-


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dichlorophenyl)ethanol could be used to synthesize luliconazole (antifungal drug).7 Considering their great value for synthetic applications, recently, several efforts have been tried to prepare chiral halohydrins using ketoreductases.8-12 However, the wild-type enzymes usually display insufficient activity or selectivity, which is difficult to meet industrial demands. Furthermore, most ketoreductases would generate (R)-halohydrins since these enzymes mainly follow the Prelog’s rule and ketoreductases with anti-Prelog stereoselectivity are relatively rare in nature.8 Thus, the discovery of new ketoreductases with efficient catalytic properties or engineering of enzymes to desired functions are still needed. Ketoreductase 1 obtained from Candida glabrata (CgKR1) could catalyze the reduction of various ketoesters to corresponding chiral alcohols at high substrate concentration with excellent stereoselectivity,13,14 such as (R)-o-chloromandelate and ethyl (R)-2-hydroxy-4-phenylbutyrate, which are the key intermediates for synthesis of clopidogrel and angiotensin-converting enzyme (ACE) inhibitors.13,14 Our previous research also revealed that CgKR1 displays high stereoselectivity towards para-, meta- and ortho-substituted acetophenones.15 Interestingly, CgKR1 catalyzes para-, meta-halogen substituted acetophenones following the Prelog’s rule while the reduction of ortho-halogen substituted acetophenones follows the anti-Prelog’s rule.15 However, CgKR1 displays low activity and selectivity towards α-halogen-substituted acetophenones, which could yield important building blocks halohydrins. Herein, we reported the engineering of CgKR1 towards 2-chloro-acetophenone (1a, Scheme 1), 2-chloro-4'fluoroacetophenone (2a), 2-bromo-acetophenone (3a) and other substrates (4a-7a). Through test minimum mutants at residues Phe92 and Tyr208, evolved ketoreductases with high stereoselectivity towards 2-chloro-acetophenone, 2-bromo-acetophenone and also other substrates were obtained. We also made whole-cell biocatalysts that could accept high


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concentration substrates by co-expression of CgKR1 mutants and glucose dehydrogenase (GDH) from Bacillus subtilis.

RESULTS AND DISCUSSION To shed light on the structure and molecular basis for engineering of CgKR1, we first solved the crystal structure of apo-state CgKR1 at 1.7 Å resolution. The structural comparison using Dali server16 revealed that the overall structure of CgKR1 shows high similarity to yeast methylglyoxal/isovaleraldehyde reductase Gre2 (RMSD = 1.4 Å for the Cα atoms; PDB ID: 4PVC),17 which has 61% sequence identity to CgKR1 (Figure 1a). Moreover, the catalytic residues (Ser134, Tyr175, Lys179) of CgKR1 are nearly identical to corresponding residues in the structure of Gre2 (Figure 1b). In case of NADPH binding residues, most of them in CgKR1 show quite similar architectures to corresponding residues in Gre2, except Thr16 and Ser226 (Figure S1 in the Supporting Information). As reported, the NADPH binding of Gre2 or related ketoreductases would induce a significant conformational change from an open form to a closed form of enzyme,17 which probably facilitate the binding of substrates. So, we modeled and optimized the structure of CgKR1 with closed conformation based on the crystal structure of NADPH-complex Gre2 (PDB ID: 4PVD). Since CgKR1 and Gre2 have high sequence identities and crystal structure similarities (apo form), we expect the modeled structure to be useful and sufficient as a basis for further analyses. To better understand the substrates recognition of CgKR1 and to choose appropriate residues for engineering, 2-chloro-acetophenone (1a) was docked into the modeled structure of CgKR1 using AutoDock Vina program.18 As shown in Figure 1c, compound 1a is located in a reasonable


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conformation in which the carbonyl group points towards catalytic Ser134 and Tyr175, while the carbonyl carbon atom of 1a is in probable position for the hydride transfer from a NADPH molecule. Further analyses of the model reveal that the chloromethyl of 1a is placed in a small binding pocket created by residues Phe92, Phe94, Ile172, and Tyr175, while the phenyl group of 1a is surrounded by a large binding pocket created by Ser134, Phe135, Ala136, Pro206, Val207 and Tyr208 (Figure 1c-d). This conformation would lead the production of (R)-2-chloro-1phenylethanol ((R)-1b) by following Prelog’s rule, that is consistent with the experiment results. By comparing the structures of CgKR1 with other ketoreductases that have similar sequences, we notice that the bulky Phe92, a residue in a flexible loop (residues 89–98) of CgKR1, prevents the binding of large group of substrates in the small binding pocket (Figure 1c-d). Mutations of Phe92 to smaller residues, such as Ala, Ile or Val, may reshape and enlarge the potential small binding pocket for substrates. Moreover, it has been reported that the flexible loop that contains Phe85 of Gre2 (corresponded to Phe92 of CgKR1, Figure 1c) shifts toward NADPH after binding cofactor,17 indicating that corresponding loop in CgKR1 is crucial for substrate recognition. Notably, this loop in the crystal structure of apo-CgKR1 was disordered since the density of residues 94 and 95 were missed. Although not fully solved, the direction of this loop in apo-CgKR1 was different from apo-Gre2 structure (Figure S1 in the Supporting Information). These results also suggested that this flexible loop in CgKR1 is important for substrate positioning (and stereoselectivity) and the conformation should occur after binding of NADPH in the active site. With the evidence from crystal structure and docking simulation, we proposed that mutations of Phe92 in this loop of CgKR1 might influence the substrate binding and thereby alter the catalytic efficiency and selectivity. Therefore, Phe92 was first selected for engineering.


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Instead of saturation mutagenesis (SM) to other 19 residues or NDC/NDT-based SM, which have been successfully applied for protein engineering of several enzymes by different groups1922

and also our group23, point mutagenesis to 6 smaller residues (Ala, Val, Ile, Ser, Thr and Leu)

were constructed for Phe92 of CgKR1. Asymmetric reduction of ketones (1a-7a) by these mutants were performed using the WT CgKR1 as control under the same reaction condition. Fortunately, as shown in Table 1, the F92A variant displayed >99% stereoselectivity towards 1a compared to the 23.1% ee of WT. The other variants also showed medium to high (21%-97%) stereoselectivity. Moreover, all the variants exhibited inversed (S)-preference while the WT exhibited (R)-preference towards 1a. These results indicated that the benzyl group of 1a is now placed in the small binding pocket created by Phe92Ala (or Val, Ile, Ser, Thr, Leu) substitution, which promotes the anti-Prelog conformation, whereas the small binding pocket created by Phe92 in the WT CgKR1 is narrow and only suitable for the chloromethyl group of 1a to proceed Prelog conformation. Asymmetric reduction of 2-chloro-4'-fluoroacetophenone (2a), para-fluoro substituted 1a, by these variants also displayed the inversed (S)-preference with 27%-94% ee values. In case of 2-bromo-acetophenone (3a), a substrate not accepted by WT, all variants gave (S)-product with 55%-95% ee values. The anti-Prelog stereopreference of these variants towards 3a was also reasonable since the halogens Br and Cl have similar molecular volumes and molecular electrostatic potentials. In contrast to 1a-3a, most of these variants catalyzed the asymmetric reduction of 4a and 5a following the Prelog’s rule, yielding (R)-4b and (S)-5b respectively (Table 1). The differences between OH group (or CH3 group) and halo groups in the substrates might cause the different stereopreferences of CgKR1 variants. In particular, excellent stereoselectivity was found in the reduction of 4a by F92V variant, which gave (R)-product with 99.2% ee value. In case of 3-


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chloro-propiophenone (6a), five variants gave (R)-alcohol following anti-Prelog’s rule, which is similar as the reduction of 1a-3a by these variants. For compound 7a, three variants gave (S)product while two variants gave (R)-product. The ortho-chloro substituent might lead to the adoption of reactive pro-S orientation or pro-R orientation of 7a in the binding pocket of different variants. Although evolved CgKR1 with excellent activity (>99% conversion) and stereoselectivity (>99% ee) towards 1a or 4a were obtained, further mutagenesis was needed for asymmetric reduction of other substrates. So, we moved attention to Tyr208, a bulky residue in the large binding pocket that accommodates phenyl groups of substrates (Figure 1d). Mutations at this position might reconfigure the space in the large binding pocket, which may lead to the more selective orientations of substrates. Thus, we first tested the single mutations of Tyr208, which was mutated to smaller residues Ala, Val, Ile, Ser, Thr or Leu as Phe92. The activity assays showed that all of these variants didn’t display any reductive activity for 3a-7a (Table 2). These results are reasonable since WT CgKR1 couldn’t reduce these substrates neither. Despite this, some variants still displayed more stereoselectivity towards 1a and 2a compared to the WT CgKR1, which inspire us the potential of double mutations at both Phe92 and Tyr208 residues. Then we selected F92V variant as the parent for the next mutations at position Tyr208 of CgKR1, since F92V displayed high activity. As shown in Table 3, these variants catalyzed the asymmetric reduction of 1a-5a to corresponding products with almost same configurations as the products of F92V variant. Remarkably, variants that showed high stereoselectivity and activity were obtained. For instance, the F92V/Y208T variant gave (S)-2b with 99.2% ee and >99% conversion, whereas the WT and F92V variant gave products with 32.6% ee (R) and 72.6% ee (S) values, respectively. Excellent anti-Prelog stereoselectivity was also found in the reduction of


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3a by mutant F92V/Y208I, which produced corresponding (S)-alcohol with ee value of 99.5%, while F92V gave (S)-product with 59.0% ee value. For reduction of substrate 5a, the ee value of (S)-product was enhanced from 57.1% (by F92V variant) to 97.5% (by F92V/Y208L variant). In order to investigate the importance of the F92 and Y208 substitutions for the activity, we conducted kinetic studies with CgKR1 WT and some variants towards different substrates. As shown in Table 4, for substrate 1a, the F92A variant exhibited about 400 fold enhanced kcat/Km value compared to the WT enzyme. In case of substrate 2a, the kcat/Km value of WT was also remarkably improved by F92V/Y208T variant. The kinetics parameters for WT-catalyzed reductions 3a-5a were not detected since WT enzyme didn’t show any activity towards these substrates. However, the F92V/Y208I, F92V and F92V/Y208L variants displayed high activity towards substrates 3a, 4a and 5a respectively. These results indicated that the substitutions at F92 and Y208 of CgKR1 influence both stereoselectivity and activity for the asymmetric reductions of compounds 1a-5a. To throw some light on the origins of stereoselectivity in the asymmetric reduction by evolved CgKR1, the substrates 1a were docked into the structure of F92A variant, and also the structure of WT enzyme. The structure of F92A variant was obtained in silico by mutation of WT enzyme in the target position. The docking was performed using AutoDock Vina program18 and related lowest-energy docked conformations are shown in Figure 2. In the WT enzyme (Figure 2a), the phenyl group of Phe92 sterically hindered the small binding pocket, disfavoring the orientation of the chloromethyl group of 1a in this region of the active site. This unfavorable pro-R orientation of 1a lead to the low stereoselectivity in the asymmetric reaction. In contrast, the F92A substituent remarkably enhanced the space in the small binding pocket (Figure 2b). As a result, the phenyl group of substrate 1a is located in the small pocket, while the chloromethyl


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group is placed in the large pocket. In this way, the reactive pro-S orientation of 1a in the substrate binding pocket of F92A variant gives the inversed (S)-alcohol as product following anti-Prelog’s rule. These results led us to hypothesize that mutagenesis of F92 to Y (tyrosine) or W (tryptophan) might improve the moderate R-selectivity of WT CgKR1, since the bulky substitutions would decrease the small binding pocket that accommodates the chloromethyl group of 1a. Thus F92Y and F92W variants were constructed and tested. As shown in Table S4 in the Supporting Information, although with lower activity, the R-selectivity of F92Y and F92W variants towards 1a were improved to 33.6% and 94.7% respectively, compared to the 23.1% ee value of WT CgKR1. These additional evidences supported the explains for anti-Prelog-selective variants such as F92A and also raised the potentials of further engineering of CgKR1 to Prelogselective catalyst towards α-halo ketones. Although the evolved ketoreductases with high stereoselectivity (>99% ee) and activity (>99% conversion) towards α-halo ketones 1a-3a were obtained, these biocatalysts were not yet of practical utility. To test their potential in scale up-reactions, we first constructed the whole cell biocatalysts by co-expression of CgKR1 variants in Escherichia coli with glucose dehydrogenase (GDH),15 which was applied for regeneration of NADPH. The asymmetric reductions were performed using lyophilized E. coli cells. Co-expression of GDH and CgKR1 in one cell would simplify the operation procedure, which will make the biocatalytic process more practicable. Furthermore, the whole cell biocatalysts might reduce the cost of enzymatic activity in reaction using separated crude enzymes, that will enhance the tolerance to substrates and co-solvents with high concentrations. Since the solubility of the ketones 1a-3a in water are low, reactions were operated using dimethylsulfoxide (DMSO) as co-solvent and at high temperature (40 °C) to improve the solubility. After a simple optimization, F92A variant (40g L-1 lyophilized cells)


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could converted 40 g L-1 ketone 1a to corresponding (S)-alcohol of >99% ee in 10% DMSO (Table 5). Similarly, the F92V/Y208T variant could efficiently catalyze the asymmetric reduction of 2a at a 60 g L-1 loading in the presence of 10% DMSO within 4 h, with a >99% ee value (Table 5). Furthermore, the F92V/Y208I variant of CgKR1 showed high conversion (>99%) and selectivity (>99% ee) at a substrate loading of 100 g L-1 for asymmetric reduction of 3a (Table 5). The obtained results of stereoselective reduction of 2-bromo-acetophenone (3a) are valuable since that most of reported research focused on the reduction of 2-chloro-acetophenone (1a).8-10,12 Also, the space-time yield of this reaction (3a) was 600 g L-1 day-1, which was comparable to that of 1a in literature.10 These results indicated the potential of practical applications of CgKR1 variants for preparation of enantiopure halohydrins by asymmetric reduction of α-halo ketones after further study in the future.

CONCLUSION Enantiopure aryl halohydrins, which could be synthesized by biocatalytic reduction of α-halogen substituted acetophenones via ketoreductases, are important building blocks for pharmaceuticals. In this study, through minimum mutagenesis at residues Phe92 and Tyr208, which was located in the flexible loop and the binding pocket of CgKR1 respectively, evolved ketoreductases with excellent stereoselectivity (>99% ee) towards 2-chloro-acetophenone (1a), 2-chloro-4'fluoroacetophenone (2a), and 2-bromo-acetophenone (3a) were obtained, indicating that CgKR1 could be tailored for high activity and selectivity. Interestingly, the variants displayed anti-Prelog stereoselectivity towards these aromatic α-halo ketones, that would yield more valuable (S)halohydrins. In addition, biocatalytic processes in laboratory using CgKR1 variants/GDH coexpressed whole cell biocatalysts for reduction of ketones 1a-3a at medium to high substrate


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loading (40 g L-1, 60 g L-1, and 100 g L-1 for 1a, 2a, and 3a, respectively) were achieved, indicating the potential of practical applications of these variants through further study in the future.

ASSOCIATED CONTENT Supporting Information. Experimental details, supplementary tables and figures are provided. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail:[email protected]. Tel/Fax: 86-24-23986436 Present Addresses ┴

Laboratory of Organic Chemistry, ETH Zürich, 8093 Zürich, Switzerland.

Author Contributions ‡

F.Q and B.Q. contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the Science and Technology Research Projects from the Ministry of Education of the People’s Republic of China (213007A). REFERENCES


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(1) Hall, M.; Bommarius, A. S. Chem. Rev. 2011, 111, 4088-4110. (2) Monti, D.; Ottolina, G.; Carrea, G.; Riva, S. Chem. Rev. 2011, 111, 4111-4140. (3) Wildeman, S. M. D.; Sonke, T.; Schoemaker, H. E.; May, O. Acc. Chem. Res. 2007, 40, 1260-1266. (4) Bornscheuer, U.; Huisman, G.; Kazlauskas, R.; Lutz, S.; Moore, J.; Robins, K. Nature 2012, 485, 185-194. (5) Huisman, G. W.; Liang, J.; Krebber, A. Curr. Opin. Chem. Biol. 2010, 14, 122-129. (6) Gras, J., Mirabegron for the treatment of overactive bladder. Drugs Today (Barc) 2012, 48, 25-32. (7) Uchida, K.; Nishiyama, Y.; Yamaguchi, H. J. Infect. Chemother. 2004, 10, 216-219. (8) Li, A.; Ye, L.; Yang, X.; Yang, C.; Gu, J.; Yu, H. Chem. Commun. 2016, 52, 6284-6287. (9) Xu, G.; Yu, H.; Shang, Y.; Xu, J. RSC Adv. 2015, 5, 22703-22711. (10) Xu, G.; Shang, Y.; Yu, H.; Xu, J. Chem. Commun. 2015, 51, 15728-15731. (11) Wang, S.; Nie, Y.; Xu, Y.; Zhang, R.; Ko, T. P.; Huang, C. H.; Chan, H. C.; Guo, R. T.; Xiao, R. Chem. Commun. 2014, 50, 7770-7772. (12) Xu, G.; Yu, H.; Zhang, X.; Xu, J. ACS Catal. 2012, 2, 2566-2571. (13) Ma, H.; Yang, L.; Ni, Y.; Zhang, J.; Li, C. X.; Zheng, G.; Yang, H.; Xu, J. Adv. Synth. Catal. 2012, 354, 1765-1772. (14) Huang, L.; Ma, H.; Yu, H.; Xu, J. Adv. Synth. Catal. 2014, 356, 1943-1948. (15) Liang, P.; Qin, B.; Mu, M.; Zhang, X.; Jia, X.; You, S. Biotechnol. Lett. 2013, 35, 14691473. (16) Holm, L.; Rosenström, P. Nucleic Acids Res. 2010, 38, W545-W549. (17) Guo, P.; Bao, Z.; Ma, X.; Xia, Q.; Li, W. BBA-Proteins Proteom. 2014, 1844, 1486-1492.


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(18) Trott, O.; Olson, A. J. J. Comput. Chem. 2010, 31, 455-461. (19) Reetz, M. T. J. Am. Chem. Soc. 2013, 135, 12480-12496. (20) Reetz, M. T. Angew. Chem., Inter. Ed. 2011, 50, 138-174. (21) Zhang, D.; Chen, X.; Chi, J.; Feng, J.; Wu, Q.; Zhu, D. ACS Catal. 2015, 5, 2452-2457. (22) Ye, L.; Toh, H. H.; Yang, Y.; Adams, J. P.; Snajdrova, R.; Li, Z. ACS Catal. 2015, 5, 11191122. (23) Qin, B.; Liang, P.; Jia, X.; Zhang, X.; Mu, M.; Wang, X.; Ma, G.; Jin, D.; You, S. Catal. Commun. 2013, 38, 1-5.


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Scheme 1. Asymmetric reduction of ketones 1a-7a following Prelog’s rule or anti-Prelog’s rule.


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Figure 1. (a) Comparison of overall structure of apo-state CgKR1 (white) with Gre2 (orange; PDB ID: 4PVC). The coordinate and structure factor of apo-CgKR1 have been deposited in the Protein Databank with the accession code 5B6K. (b) Comparison of catalytic residues of CgKR1 (white) with Gre2 (orange; PDB ID: 4PVC). (c) Docking of 1a into the active site of CgKR1. The catalytic residues (S134, Y175 and K179) and NADPH are shown as white sticks. Residues along the small and large binding pockets are shown as blue sticks. The flexible loop is shown in green. (d) Substrate binding pocket of CgKR1. F92 in the small pocket is shown in magenta while the Y208 in the large binding pocket is shown in cyan. F92 and Y208 were selected for mutation in this study.


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Figure 2. (a) Docking of 1a into the binding pocket of WT CgKR1. The F92 (magenta) and Y208 (cyan) promote of a productive pro-R orientation of 1a (yellow). (b) Docking of 1a into the binding pocket of F92A variant. F92A enlarges the small binding pocket and leads to a productive pro-S orientation of 1a.


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Table 1. Asymmetric reduction of ketones 1a-7a by WT CgKR1 and variants at F92 position Substrate

WT

F92A

F92V

F92I

F92S

F92T

F92L

1a

72.6a, 23.1b(R)c

>99, 99.1(S)

97.4, 81.3(S)

95.2, 21.3(S)

>99, 97.1(S)

>99, 89.7(S)

>99, 75.1(S)

2a

90.1, 32.6(R)

82.5, 94.2(S)

73.4, 72.6(S)

97.3, 66.1(S)

>99, 92.0(S)

>99, 77.4(S)

>99, 27.2(S)

3a

NAd, NA

61.7, 50.5(S)

>99, 59.0(S)

96.8, 91.2(S)

64.7, 93.6(S)

56.3, 94.7(S)

81.6, 54.6(S)

4a

NA, NA

14.2, 13.4(S)

>99, 99.2(R)

66.6, 93.3(R)

23.8, 13.1(R)

96.3, 71.2(R)

81.3, 95.7(R)

5a

NA, NA

NA, NA

42.9, 57.1(S)

27.2, 61.5(S)

8.2, 42.4(R)

NA, NA

27.5, 88.9(S)

6a

NA, NA

32.7, 56.2(R)

89.5, 60.8(S)

38.1, 45.3(R)

28.3, 62.4(R)

34.2, 28.1(R)

39.1, 86.6(R)

7a

NA, NA

9.2, 84.9(S)

32.4, 50.8(R)

37.3, 3.8(R)

8.7, 75.3(S)

NA, NA

21.4, 48.5(R)

a

Conversion [%]. bThe enantiomeric excess (ee) values of resultant alcohols [%], which were measured by chiral HPLC analysis. cThe absolute configurations of resultant alcohols, which were identified by comparing the retention times of chiral HPLC with authentic samples or literature data (Table S3 in the Supporting Information). The absolute configurations of resultant alcohols from Prelog reduction were shown in green, while resultant alcohols from anti-Prelog reduction were shown in blue. dNA, no measurable activity.


18

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ACS Catalysis

Table 2. Asymmetric reduction of ketones 1a-2a by variants at Y208 position of CgKR1 Substrate

WT

Y208A

Y208V

Y208I

Y208S

Y208T

Y208L

1a

72.6, 23.1(R)

36.8, 32.6(R)

74.3, 51.8(R)

76.4, 11.5(R)

NA, NA

NA, NA

79.5, 78.8(R)

2a

90.1, 32.6(R)

42.6, 3.8(R)

80.2, 23.6(R)

81.7, 39.6(R)

10.4, 6.2(R)

11.6, 51.8(R)

84.1, 67.3(R)


19

ACS Catalysis

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Page 20 of 22

Table 3. Asymmetric reduction of ketones 1a-7a by variants at F92/Y208 positions of CgKR1

Substrate

WT

F92V/ Y208A

F92V/ Y208V

F92V/ Y208I

F92V/ Y208S

F92V/ Y208T

F92V/ Y208L

1a

72.6, 23.1(R)

>99, 90.2(S)

>99, 93.7(S)

>99, 95.3(S)

>99, 92.0(S)

>99, 98.3(S)

>99, 42.9(S)

2a

90.1, 32.6(R)

>99, 97.8(S)

>99, 98.8(S)

>99, 98.9(S)

>99, 98.6(S)

>99, 99.2(S)

>99, 65.0(S)

3a

NA, NA

>99, 94.7(S)

>99, 96.2(S)

>99, 99.5(S)

>99, 91.2(S)

>99, 93.5(S)

>99, 92.0(S)

4a

NA, NA

27.3, 94.9(R)

56.4, 95.7(R)

52.6, 97.2(R)

8.2, 95.4(R)

10.1, 87.5(R)

46.7, 97.5(R)

5a

NA, NA

73.5, 12.4(S)

92.6, 40.4(S)

97.3, 27.7(S)

23.3, 16.4(S)

28.4, 55.1(S)

>99, 97.2(S)

6a

NA, NA

98.2, 32.1(S)

>99, 7.8(R)

98.8, 24.2(R)

81.7, 31.6(S)

92.6, 41.6(S)

>99, 79.5(R)

7a

NA, NA

NA, NA

NA, NA

NA, NA

NA, NA

NA, NA

NA, NA


20

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

ACS Catalysis

Table 4. Kinetic parameters of the WT for 1a-2a and best variants for 1a-5a Enzyme

Km (mM)

kcat (min-1)

kcat/Km (mM-1 min-1)

WT

6.2 ± 0.01

0.02 ± 0.001

0.004

F92A

1.0 ± 0.01

1.7 ± 0.001

1.6

WT

7.2 ± 0.10

0.03 ± 0.01

0.005

F92V/Y208T

0.8 ± 0.01

4570.2 ± 0.24

5573.4

3a

F92V/Y208I

1.0 ± 0.01

5812.8 ± 0.14

5933.4

4a

F92V

13.2 ± 0.02

154.2 ± 0.10

11.7

5a

F92V/Y208L

5.4 ± 0.01

124.8 ± 0.16

23.2

Substrate 1a

2a


21

ACS Catalysis

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Page 22 of 22

Table 5. Asymmetric reduction of 1a-3a with high substrate loading

Substrate

1a

2a

3a

Enzyme

Concentration (g L-1)

Time (h)

Conversion (%)

ee (%)

F92A

20

2.5

>99

99.7

F92A

40

4.0

>99

99.7

F92A

60

8.0

65.9

99.7

F92V/Y208T

20

1.5

>99

99.6

F92V/Y208T

40

3.0

>99

99.6

F92V/Y208T

60

4.0

>99

99.6

F92V/Y208T

80

8.0

83.4

99.6

F92V/Y208I

20

1.0

>99

99.6

F92V/Y208I

40

1.5

>99

99.6

F92V/Y208I

60

2.5

>99

99.6

F92V/Y208I

80

3.0

>99

99.6

F92V/Y208I

100

4.0

>99

99.6

F92V/Y208I

120

8.0

83.3

99.6


22

Engineering of Candida glabrata Ketoreductase 1 for Asymmetric

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