Volume 50 Number 58 25 July 2014 Pages 7729–7906
ChemComm Chemical Communications www.rsc.org/chemcomm
ISSN 1359-7345
COMMUNICATION Yan Xu, Rey-Ting Guo et al. Unconserved substrate-binding sites direct the stereoselectivity of medium-chain alcohol dehydrogenase
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Cite this: Chem. Commun., 2014, 50, 7770 Received 8th March 2014, Accepted 30th April 2014
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Unconserved substrate-binding sites direct the stereoselectivity of medium-chain alcohol dehydrogenase† Shanshan Wang,‡a Yao Nie,‡a Yan Xu,*a Rongzhen Zhang,a Tzu-Ping Ko,b Chun-Hsiang Huang,c Hsiu-Chien Chan,c Rey-Ting Guo*c and Rong Xiaod
DOI: 10.1039/c4cc01752h www.rsc.org/chemcomm
Structure-guided design of substrate-binding pocket inversed the stereoselectivity of an NADH-dependent medium-chain alcohol dehydrogenase (MDR) from Prelog to anti-Prelog. The pocket-forming amino acids, especially the unconserved residues as hotspots, play critical roles in directing MDRs’ stereoselectivity.
Optically active alcohols play a rapidly growing role as building blocks for the synthesis of pharmaceuticals and fine chemicals. Among them, enantiopure aryl alcohols serve as valuable intermediates for preparation of antidepressants, anti-asthmatics, cholesterol-lowering agents, adrenergic receptor agonists, and NK1 antagonists, etc.1 Attractive access to the alcohols involves alcohol dehydrogenase (ADH)-mediated asymmetric reduction of ketones with unparalleled chemo-, regio-, and stereoselectivities under benign conditions.2 Concerning the stereochemical patterns of hydride transfer from NAD(P)H to ketones, ADHs perform either the Prelog or anti-Prelog stereoselectivity.3 In nature, very few ADHs follow the anti-Prelog’s rule, particularly medium-chain ADHs (MDRs).4 Furthermore, the limited antiPrelog ADHs require more expensive NADPH as a cofactor.5 Therefore, anti-Prelog NADH-dependent ADHs are extremely in demand. Previously, NADH-dependent (R)-specific carbonyl reductase (RCR) from Candida parapsilosis, an MDR member, was found to catalyze asymmetric reduction of prochiral aryl
a
School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. E-mail: [email protected]; Fax: +86-510-85864112; Tel: +86-510-85918201 b Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan c Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. E-mail: [email protected]; Fax: +86-022-84861926; Tel: +86-022-84861999 d Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, USA † Electronic supplementary information (ESI) available: Experimental section, supplementary tables and figures, and crystal structures deposited in the PDB under the accession codes: 3WLE, 3WLF and 3WNQ. See DOI: 10.1039/c4cc01752h ‡ These authors contributed equally to this work.
7770 | Chem. Commun., 2014, 50, 7770--7772
Scheme 1 Asymmetric reduction of aryl ketones (1a–8a) to the corresponding alcohols (1b–8b) by RCR using NADH as a hydrogen donor.
ketones to corresponding alcohols with excellent stereoselectivity in Prelog configuration (Scheme 1).6 To date, only human ADH aa has the opposite stereoselectivity in structure-solved stereoselective MDRs.7 Thermoanaerobacter brockii ADH (TbADH) has a substrate-size-induced reversal of stereoselectivity (Table S1, ESI†).8 Moreover, the same stereoselective enzymes bear a variety of stereopreferences towards different substrates. Thus, it was proposed that the diversity of MDRs’ stereoselectivity would be dominated by the primary structure and the cavity shape in active sites of enzymes. Recent research studies have focused on the design of stereocontrolled reactions.9 By contrast, stereoselectivity switch of MDRs is rare. So far, only the NADPH-dependent ADH from T. ethanolicus was reported to alter stereoselectivity by substituting Ala for I86 and C295, or Thr for S39.10 Although it is known that MDRs’ stereoselectivity depends on the relative sizes of two substituents neighboring the carbonyl group, a puzzling question remains on the molecular basis of enzyme stereorecognition and substrate orientation. Therefore, engineering MDRs performing anti-Prelog stereoselectivity is still a major challenge. The structural details and the stereoselective mechanism of RCR are yet unclear, although RCR shares 31.5% sequence identity with ADH-‘A’ (PDB codes: 2XAA and 3JV7).11 Herein, we solved the complex structures of RCR with a cofactor and a substrate/product (Table S2, ESI†), and performed the rational design of a Prelog NADH-dependent ADH for inversion of stereoselectivity towards aryl ketones.
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The holo-RCR with NAD+ revealed as a homo-tetramer, and each subunit was composed of a cofactor-binding domain and a catalytic domain. Between these two domains lay a NAD+ molecule. The NAD+ binding induced a significant conformational change from an open form to a closed form of the enzyme (Fig. S1–S3, ESI†), which resembles other MDRs.12 In the binary complex with (R)-1b, the molecule was bound in the hydrophobic cavity formed by residues L55, W116, L119, D154 and F285. Most importantly, the OG of S46 was in hydrogen-bonding distance to the O1 of (R)-1b. The N of W286 and the OG1 of T158 interacted with the O2 of (R)-1b by hydrogen bonds, which determined its orientation along with the hydrophobic interactions (Fig. S4, ESI†). The RCR_H49A mutant complex with 1a further confirmed its binding orientation in the substrate pocket. Based on the solved holo-RCR, the structure model of a ternary complex with NAD+ and 1a displayed a hydrophobic substratebinding pocket, matching the observation that this enzyme was adapted to accept aryl ketones.6 The substrate-binding cavity can be divided into a large and a small pocket (Fig. 1A). The large pocket is composed of S46, H49, V50, L55, C57, H65, W116, L119 and L262, while the small pocket is formed by C44, D154, T158, F285 and W286. The architecture of pockets allows the phenyl ring of 1a to fit into the large pocket, and the hydroxymethyl group to enter the small pocket. Consequently, the orientation of 1a ensures that NADH delivers its pro-R hydride to the re-face of 1a to produce (R)-1b, following the Prelog’s rule (Fig. S5, ESI†). Although every MDR has its unique substrate-binding pocket, and possesses versatile stereoselectivity and function, one question is how the amino acid composition determines the pocket shape and controls the stereoselectivity and stereopreference. Combined with other solved stereoselective MDR structures, we are interested in the pocket-forming amino acids (Fig. S6, ESI†). The residues equivalent to S46, H49, V50 and H65 lining the large pocket of RCR are relatively conserved in MDRs, with H49 and H65 nearly invariant. However, the positions at L55, C57, W116, L119 and L262 are varied. Moreover, in the small pocket, the C44, D154 and T158 are relatively conserved, while at the F285 and W286 are optional (Fig. 1B). These varied residues might lead to the different substrate-binding pockets and stereopreferences of MDRs.
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At present, only one structure of anti-Prelog MDR, human ADH aa, has been determined, in which the T48 and A93 are key residues contributing to the anti-Prelog stereoselectivity.7,13 Compared to isoenzyme aa, S46 in RCR with the smaller side chain than T48 lines the large pocket. Interestingly, although G91 in RCR (corresponding to A93 in isoenzyme aa F93 in HLADH) has no side chain, the bulky side chain of W286 as an important residue like F93 in HLADH forms the small pocket, in agreement with the same Prelog stereoselectivity of HLADH (Fig. S6, ESI†). In addition, RCR had more bulky side chains at F285 and W286, located at the entrance of and inside of the small pocket, respectively, than those of other MDRs, such as I-F in HLADH, L-V in SsADH, I-V in PaADH and CbADH (Fig. S7, ESI†). So we considered that the F285 and W286 might play critical roles. It is hypothesized that non-conservative substitutions with Ala at the positions would greatly enlarge the small pocket and affect the enantiomeric specificity of RCR. Three variants of F285A, W286A and F285A/W286A were constructed (Table S3, ESI†) and examined in asymmetric reduction of aryl ketones (1a–8a). As expected, the variants indeed changed the stereoselectivity towards the substrates. It is likely that stepwise enlarged small pocket from the wide type (WT) to the double-mutant F285A/W286A led to a stepwise change in stereoselectivity and stereopreference towards 2a and 4a–8a. Exceptionally, all three variants completely inversed the enantioselectivity towards 3a to the (S)-product with 499.9% ee. F285A/W286A catalyzed all substrates with the opposite enantioselectivity, exhibiting high enantioselectivity towards 3a and good enantioselectivity towards 1a, 4a and 6a. W286A also successfully inverted the enantioselectivity towards 2a–4a and 6a, and F285A lost most of the original enantioselectivity and contributed to double-mutant F285A/W286A with the inverted enantioselectivity (Table 1). The ee values of the desired enantiomer products might not be all ideal for practical utility, whereas the landmark results will serve as a starting point for further protein engineering of RCR and other related enzymes. To understand the molecular basis of the altered stereoselectivity and stereopreference, we performed automated docking experiments. Models of variants with the substrate 1a bound to the active sites showed that a reasonable docking conformation in F285A yielded the (R)-enantiomer, while two opposite conformations in W286A produced (R)- and (S)-enantiomers (re-face orientation still had slightly higher affinity than that of si-face), which was qualitatively consistent with the maintained enantioselectivity of F285A and
Table 1
Fig. 1 The substrate-binding pocket of RCR. (A) Surface of the large pocket of the phenyl-binding site (blue) and the small pocket of the hydroxymethylbinding site (green). NAD+ (yellow) and 1a (cyan) are shown as thick sticks. (B) The pocket-forming residues are shown as thin sticks. The varied residues (magentas and orange) and the conserved residues (blue and green) in the large and the small pocket. The zinc ion (gray) is shown as a sphere.
This journal is © The Royal Society of Chemistry 2014
Enantioselectivity for RCR WT and variants towards aryl ketonesa
Substrate
WT
1a 2a 3a 4a 5a 6a 7a 8a
499.9 499.9 499.9 97.3 73.8 499.9 80.6 499.9
a
F285A (R) (R) (R) (S) (S) (S) (S) (S)
499.9 59.4 499.9 35.1 24.4 71.9 48.9 71.7
W286A (R) (R) (S) (S) (S) (S) (S) (S)
77.3 35.1 499.9 57.4 23.3 52.4 8.1 64.6
F285A/W286A (R) (S) (S) (R) (S) (R) (R) (S)
73.3 47.5 499.9 85.6 15.3 76.1 63.6 21.6
(S) (S) (S) (R) (R) (R) (R) (R)
Determination of enantiomeric excess by HPLC analysis (Fig. S8–S15, ESI).
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Fig. 2 Comparison of docking the substrates 1a (A), 3a (B) and 4a (C) in the active sites of (a) F285A (blue), (b) W286A (orange and green, the affinity of orange conformation higher than that of the green conformation) and (c) F285A/W286A (cyan) with WT (magentas).
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difference in pocket shape and substrate orientation, and thus direct the diversity of reaction stereoselectivity and stereopreference. It is probable that MDRs, even members with unknown structure, can be engineered by varying these sites to create more stereocomplementary features for production of desired optically active alcohols for the synthesis of pharmaceuticals and fine chemicals. This work was financially supported by the National Key Basic Research and Development Program of China (2011CB710800), the National Hi-Tech Research and Development Program of China (2011AA02A209, 2011AA02A210), the National Natural Science Foundation of China (21336009, 21376107), the Program of Introducing Talents of Discipline to Universities (111-2-06), the High-End Foreign Experts Recruitment Program (GDW20133200113), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. We thank the National Synchrotron Radiation Research Center of Taiwan for beamtime allocation and data-collection assistance.
Notes and references the impaired enantioselectivity of W286A. In addition, the conformation in F285A/W286A was completely inverted, leading to a hydride transfer from pro-R hydrogen of the C4N cofactor to the si-face of 1a (Fig. 2A). The conformations of 4a further reflected a tendency from re-face orientation of WT to si-face orientation of the double variant, showing the inverted enantioselectivity of W286A and F285A/W286A (Fig. 2C). Given the excitingly inversed preference towards 3a, in contrast to WT, the opposite fit of 3a in all three variants allowed the cofactor to deliver its hydride to the si-face of 3a, instead of the re-face, producing (S)-alcohol (Fig. 2B). It is demonstrated that introducing Ala rather than bulky aliphatic residues (Leu, Ile or Val) adequately enlarged the small pocket to accommodate the large group of aryl substrates and create the anti-Prelog enantioselectivity. Furthermore, the affinity and catalytic properties of the variants for 1a–8a were altered to a varying degree. W286A and F285A/ W286A exhibited much lower Km values and higher kcat/Km values towards substrates containing substituents on the phenyl ring with respect to WT, such as 3a, 5a–7a (Table S4, ESI†). The enhanced catalytic efficiency could be attributed to the enlarged pocket of variants, which better accommodated the bulky substrates. In summary, we solved the complex structures of RCR with a cofactor and a substrate/product. Combined with other known stereoselective MDR structures, structure-guided prediction for creation of the anti-Prelog NADH-dependent feature was performed. The functional experiments of variants W286A and F285A/W286A revealed that the substrate orientation was changed and the stereoselectivity was inversed towards aryl ketones. Moreover, the variants eliminated steric repulsion with bulky substituent groups on the phenyl ring and accepted more space-demanding substrates. It becomes clear now that the cavity-forming amino acids, especially the unconserved residues as ‘‘hotspots’’, may contribute to the
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1 E. Garcı´a-Urdiales, I. Alfonso and V. Gotor, Chem. Rev., 2011, 111, 110–180; Y. Huang, N. Liu, X. R. Wu and Y. J. Chen, Curr. Org. Chem., ¨lsdorf, H. Liu and 2010, 14, 1447–1460; A. Dennig, N. Lu U. Schwaneberg, Angew. Chem., Int. Ed., 2013, 52, 8459–8462. 2 T. Matsuda, R. Yamanaka and K. Nakamura, Tetrahedron: Asymmetry, 2009, 20, 513–557; R. Wohlgemuth, Curr. Opin. Microbiol., 2010, 13, 283–292; M. M. Musa and R. S. Phillips, Catal. Sci. Technol., 2011, 1, 1311–1323. 3 V. Prelog, Pure Appl. Chem., 1964, 9, 119–130. 4 W. Kroutil, H. Mang, K. Edegger and K. Faber, Curr. Opin. Chem. Biol., 2004, 8, 120–126; S. M. A. D. Wildeman, T. Sonke, H. E. Schoemaker and O. May, Acc. Chem. Res., 2007, 40, 1260–1266. 5 C. Rodriguez, W. Borzecka, J. H. Sattler, W. Kroutil, I. Lavandera and V. Gotor, Org. Biomol. Chem., 2014, 12, 673–681; A. Weckbecker and W. Hummel, Biocatal. Biotransform., 2006, 24, 380–389. 6 Y. Nie, Y. Xu and X. Q. Mu, Org. Process Res. Dev., 2004, 8, 246–251; Y. Nie, Y. Xu, M. Yang and X. Q. Mu, Lett. Appl. Microbiol., 2007, 44, 555–562. 7 B. J. Gibbons and T. D. Hurley, Biochemistry, 2004, 43, 12555–12562. 8 C. Heiss, M. Laivenieks, J. G. Zeikus and R. S. Phillips, J. Am. Chem. Soc., 2001, 123, 345–346. 9 S. Kille, F. E. Zilly, J. P. Acevedo and M. T. Reetz, Nat. Chem., 2011, 3, 738–743; S. M. Pratter, C. Konstantinovics, C. L. M. DiGiuro, E. Leitner, D. Kumar, S. P. de Visser, G. Grogan and G. D. Straganz, Angew. Chem., Int. Ed., 2013, 125, 9859–9863; A. Shehzad, S. Panneerselvam, M. Linow, M. Bocola, D. Roccatano, J. Mueller-Dieckmann, M. Wilmanns and U. Schwaneberg, Chem. Commun., 2013, 49, 4694–4696; W. L. Tang, Z. Li and H. M. Zhao, Chem. Commun., 2010, 46, 5461–5463. 10 M. M. Musa, N. Lott, M. Laivenieks, L. Watanabe, C. Vieille and R. S. Phillips, ChemCatChem, 2009, 1, 89–93; A. E. Tripp, D. S. Burdette, J. G. Zeikus and R. S. Phillips, J. Am. Chem. Soc., 1998, 120, 5137–5141; C. Heiss, M. Laivenieks, J. G. Zeikus and R. S. Phillips, Bioorg. Med. Chem., 2001, 9, 1659–1666. 11 M. Karabec, A. Łyskowski, K. C. Tauber, G. Steinkellner, W. Kroutil, G. Grogan and K. Gruber, Chem. Commun., 2010, 46, 6314–6316. 12 B. V. Plapp, Arch. Biochem. Biophys., 2010, 493, 3–12; C. Kang, R. Hayes, E. J. Sanchez, B. N. Webb, Q. Li, T. Hooper, M. S. Nissen and L. Xun, Mol. Microbiol., 2012, 83, 85–95. 13 D. W. Green, H.-W. Sun and B. V. Plapp, J. Biol. Chem., 1993, 268, 7792–7798.
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ChemComm Chemical Communications www.rsc.org/chemcomm
ISSN 1359-7345
COMMUNICATION Yan Xu, Rey-Ting Guo et al. Unconserved substrate-binding sites direct the stereoselectivity of medium-chain alcohol dehydrogenase
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Cite this: Chem. Commun., 2014, 50, 7770 Received 8th March 2014, Accepted 30th April 2014
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Unconserved substrate-binding sites direct the stereoselectivity of medium-chain alcohol dehydrogenase† Shanshan Wang,‡a Yao Nie,‡a Yan Xu,*a Rongzhen Zhang,a Tzu-Ping Ko,b Chun-Hsiang Huang,c Hsiu-Chien Chan,c Rey-Ting Guo*c and Rong Xiaod
DOI: 10.1039/c4cc01752h www.rsc.org/chemcomm
Structure-guided design of substrate-binding pocket inversed the stereoselectivity of an NADH-dependent medium-chain alcohol dehydrogenase (MDR) from Prelog to anti-Prelog. The pocket-forming amino acids, especially the unconserved residues as hotspots, play critical roles in directing MDRs’ stereoselectivity.
Optically active alcohols play a rapidly growing role as building blocks for the synthesis of pharmaceuticals and fine chemicals. Among them, enantiopure aryl alcohols serve as valuable intermediates for preparation of antidepressants, anti-asthmatics, cholesterol-lowering agents, adrenergic receptor agonists, and NK1 antagonists, etc.1 Attractive access to the alcohols involves alcohol dehydrogenase (ADH)-mediated asymmetric reduction of ketones with unparalleled chemo-, regio-, and stereoselectivities under benign conditions.2 Concerning the stereochemical patterns of hydride transfer from NAD(P)H to ketones, ADHs perform either the Prelog or anti-Prelog stereoselectivity.3 In nature, very few ADHs follow the anti-Prelog’s rule, particularly medium-chain ADHs (MDRs).4 Furthermore, the limited antiPrelog ADHs require more expensive NADPH as a cofactor.5 Therefore, anti-Prelog NADH-dependent ADHs are extremely in demand. Previously, NADH-dependent (R)-specific carbonyl reductase (RCR) from Candida parapsilosis, an MDR member, was found to catalyze asymmetric reduction of prochiral aryl
a
School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. E-mail: [email protected]; Fax: +86-510-85864112; Tel: +86-510-85918201 b Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan c Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. E-mail: [email protected]; Fax: +86-022-84861926; Tel: +86-022-84861999 d Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, USA † Electronic supplementary information (ESI) available: Experimental section, supplementary tables and figures, and crystal structures deposited in the PDB under the accession codes: 3WLE, 3WLF and 3WNQ. See DOI: 10.1039/c4cc01752h ‡ These authors contributed equally to this work.
7770 | Chem. Commun., 2014, 50, 7770--7772
Scheme 1 Asymmetric reduction of aryl ketones (1a–8a) to the corresponding alcohols (1b–8b) by RCR using NADH as a hydrogen donor.
ketones to corresponding alcohols with excellent stereoselectivity in Prelog configuration (Scheme 1).6 To date, only human ADH aa has the opposite stereoselectivity in structure-solved stereoselective MDRs.7 Thermoanaerobacter brockii ADH (TbADH) has a substrate-size-induced reversal of stereoselectivity (Table S1, ESI†).8 Moreover, the same stereoselective enzymes bear a variety of stereopreferences towards different substrates. Thus, it was proposed that the diversity of MDRs’ stereoselectivity would be dominated by the primary structure and the cavity shape in active sites of enzymes. Recent research studies have focused on the design of stereocontrolled reactions.9 By contrast, stereoselectivity switch of MDRs is rare. So far, only the NADPH-dependent ADH from T. ethanolicus was reported to alter stereoselectivity by substituting Ala for I86 and C295, or Thr for S39.10 Although it is known that MDRs’ stereoselectivity depends on the relative sizes of two substituents neighboring the carbonyl group, a puzzling question remains on the molecular basis of enzyme stereorecognition and substrate orientation. Therefore, engineering MDRs performing anti-Prelog stereoselectivity is still a major challenge. The structural details and the stereoselective mechanism of RCR are yet unclear, although RCR shares 31.5% sequence identity with ADH-‘A’ (PDB codes: 2XAA and 3JV7).11 Herein, we solved the complex structures of RCR with a cofactor and a substrate/product (Table S2, ESI†), and performed the rational design of a Prelog NADH-dependent ADH for inversion of stereoselectivity towards aryl ketones.
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The holo-RCR with NAD+ revealed as a homo-tetramer, and each subunit was composed of a cofactor-binding domain and a catalytic domain. Between these two domains lay a NAD+ molecule. The NAD+ binding induced a significant conformational change from an open form to a closed form of the enzyme (Fig. S1–S3, ESI†), which resembles other MDRs.12 In the binary complex with (R)-1b, the molecule was bound in the hydrophobic cavity formed by residues L55, W116, L119, D154 and F285. Most importantly, the OG of S46 was in hydrogen-bonding distance to the O1 of (R)-1b. The N of W286 and the OG1 of T158 interacted with the O2 of (R)-1b by hydrogen bonds, which determined its orientation along with the hydrophobic interactions (Fig. S4, ESI†). The RCR_H49A mutant complex with 1a further confirmed its binding orientation in the substrate pocket. Based on the solved holo-RCR, the structure model of a ternary complex with NAD+ and 1a displayed a hydrophobic substratebinding pocket, matching the observation that this enzyme was adapted to accept aryl ketones.6 The substrate-binding cavity can be divided into a large and a small pocket (Fig. 1A). The large pocket is composed of S46, H49, V50, L55, C57, H65, W116, L119 and L262, while the small pocket is formed by C44, D154, T158, F285 and W286. The architecture of pockets allows the phenyl ring of 1a to fit into the large pocket, and the hydroxymethyl group to enter the small pocket. Consequently, the orientation of 1a ensures that NADH delivers its pro-R hydride to the re-face of 1a to produce (R)-1b, following the Prelog’s rule (Fig. S5, ESI†). Although every MDR has its unique substrate-binding pocket, and possesses versatile stereoselectivity and function, one question is how the amino acid composition determines the pocket shape and controls the stereoselectivity and stereopreference. Combined with other solved stereoselective MDR structures, we are interested in the pocket-forming amino acids (Fig. S6, ESI†). The residues equivalent to S46, H49, V50 and H65 lining the large pocket of RCR are relatively conserved in MDRs, with H49 and H65 nearly invariant. However, the positions at L55, C57, W116, L119 and L262 are varied. Moreover, in the small pocket, the C44, D154 and T158 are relatively conserved, while at the F285 and W286 are optional (Fig. 1B). These varied residues might lead to the different substrate-binding pockets and stereopreferences of MDRs.
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At present, only one structure of anti-Prelog MDR, human ADH aa, has been determined, in which the T48 and A93 are key residues contributing to the anti-Prelog stereoselectivity.7,13 Compared to isoenzyme aa, S46 in RCR with the smaller side chain than T48 lines the large pocket. Interestingly, although G91 in RCR (corresponding to A93 in isoenzyme aa F93 in HLADH) has no side chain, the bulky side chain of W286 as an important residue like F93 in HLADH forms the small pocket, in agreement with the same Prelog stereoselectivity of HLADH (Fig. S6, ESI†). In addition, RCR had more bulky side chains at F285 and W286, located at the entrance of and inside of the small pocket, respectively, than those of other MDRs, such as I-F in HLADH, L-V in SsADH, I-V in PaADH and CbADH (Fig. S7, ESI†). So we considered that the F285 and W286 might play critical roles. It is hypothesized that non-conservative substitutions with Ala at the positions would greatly enlarge the small pocket and affect the enantiomeric specificity of RCR. Three variants of F285A, W286A and F285A/W286A were constructed (Table S3, ESI†) and examined in asymmetric reduction of aryl ketones (1a–8a). As expected, the variants indeed changed the stereoselectivity towards the substrates. It is likely that stepwise enlarged small pocket from the wide type (WT) to the double-mutant F285A/W286A led to a stepwise change in stereoselectivity and stereopreference towards 2a and 4a–8a. Exceptionally, all three variants completely inversed the enantioselectivity towards 3a to the (S)-product with 499.9% ee. F285A/W286A catalyzed all substrates with the opposite enantioselectivity, exhibiting high enantioselectivity towards 3a and good enantioselectivity towards 1a, 4a and 6a. W286A also successfully inverted the enantioselectivity towards 2a–4a and 6a, and F285A lost most of the original enantioselectivity and contributed to double-mutant F285A/W286A with the inverted enantioselectivity (Table 1). The ee values of the desired enantiomer products might not be all ideal for practical utility, whereas the landmark results will serve as a starting point for further protein engineering of RCR and other related enzymes. To understand the molecular basis of the altered stereoselectivity and stereopreference, we performed automated docking experiments. Models of variants with the substrate 1a bound to the active sites showed that a reasonable docking conformation in F285A yielded the (R)-enantiomer, while two opposite conformations in W286A produced (R)- and (S)-enantiomers (re-face orientation still had slightly higher affinity than that of si-face), which was qualitatively consistent with the maintained enantioselectivity of F285A and
Table 1
Fig. 1 The substrate-binding pocket of RCR. (A) Surface of the large pocket of the phenyl-binding site (blue) and the small pocket of the hydroxymethylbinding site (green). NAD+ (yellow) and 1a (cyan) are shown as thick sticks. (B) The pocket-forming residues are shown as thin sticks. The varied residues (magentas and orange) and the conserved residues (blue and green) in the large and the small pocket. The zinc ion (gray) is shown as a sphere.
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Enantioselectivity for RCR WT and variants towards aryl ketonesa
Substrate
WT
1a 2a 3a 4a 5a 6a 7a 8a
499.9 499.9 499.9 97.3 73.8 499.9 80.6 499.9
a
F285A (R) (R) (R) (S) (S) (S) (S) (S)
499.9 59.4 499.9 35.1 24.4 71.9 48.9 71.7
W286A (R) (R) (S) (S) (S) (S) (S) (S)
77.3 35.1 499.9 57.4 23.3 52.4 8.1 64.6
F285A/W286A (R) (S) (S) (R) (S) (R) (R) (S)
73.3 47.5 499.9 85.6 15.3 76.1 63.6 21.6
(S) (S) (S) (R) (R) (R) (R) (R)
Determination of enantiomeric excess by HPLC analysis (Fig. S8–S15, ESI).
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Fig. 2 Comparison of docking the substrates 1a (A), 3a (B) and 4a (C) in the active sites of (a) F285A (blue), (b) W286A (orange and green, the affinity of orange conformation higher than that of the green conformation) and (c) F285A/W286A (cyan) with WT (magentas).
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difference in pocket shape and substrate orientation, and thus direct the diversity of reaction stereoselectivity and stereopreference. It is probable that MDRs, even members with unknown structure, can be engineered by varying these sites to create more stereocomplementary features for production of desired optically active alcohols for the synthesis of pharmaceuticals and fine chemicals. This work was financially supported by the National Key Basic Research and Development Program of China (2011CB710800), the National Hi-Tech Research and Development Program of China (2011AA02A209, 2011AA02A210), the National Natural Science Foundation of China (21336009, 21376107), the Program of Introducing Talents of Discipline to Universities (111-2-06), the High-End Foreign Experts Recruitment Program (GDW20133200113), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. We thank the National Synchrotron Radiation Research Center of Taiwan for beamtime allocation and data-collection assistance.
Notes and references the impaired enantioselectivity of W286A. In addition, the conformation in F285A/W286A was completely inverted, leading to a hydride transfer from pro-R hydrogen of the C4N cofactor to the si-face of 1a (Fig. 2A). The conformations of 4a further reflected a tendency from re-face orientation of WT to si-face orientation of the double variant, showing the inverted enantioselectivity of W286A and F285A/W286A (Fig. 2C). Given the excitingly inversed preference towards 3a, in contrast to WT, the opposite fit of 3a in all three variants allowed the cofactor to deliver its hydride to the si-face of 3a, instead of the re-face, producing (S)-alcohol (Fig. 2B). It is demonstrated that introducing Ala rather than bulky aliphatic residues (Leu, Ile or Val) adequately enlarged the small pocket to accommodate the large group of aryl substrates and create the anti-Prelog enantioselectivity. Furthermore, the affinity and catalytic properties of the variants for 1a–8a were altered to a varying degree. W286A and F285A/ W286A exhibited much lower Km values and higher kcat/Km values towards substrates containing substituents on the phenyl ring with respect to WT, such as 3a, 5a–7a (Table S4, ESI†). The enhanced catalytic efficiency could be attributed to the enlarged pocket of variants, which better accommodated the bulky substrates. In summary, we solved the complex structures of RCR with a cofactor and a substrate/product. Combined with other known stereoselective MDR structures, structure-guided prediction for creation of the anti-Prelog NADH-dependent feature was performed. The functional experiments of variants W286A and F285A/W286A revealed that the substrate orientation was changed and the stereoselectivity was inversed towards aryl ketones. Moreover, the variants eliminated steric repulsion with bulky substituent groups on the phenyl ring and accepted more space-demanding substrates. It becomes clear now that the cavity-forming amino acids, especially the unconserved residues as ‘‘hotspots’’, may contribute to the
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