Enzyme Engineering Based on X-ray Structures and Kinetic Profiling

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Enzyme engineering based on X-ray structures and kinetic profiling of substrate libraries: alcohol dehydrogenases for stereospecific synthesis of a broad range of chiral alcohols Yao Nie, Shanshan Wang, Yan Xu, Shenggan Luo, Yi-Lei Zhao, Rong Xiao, Gaetano Montelione, John F. Hunt, and Thomas Szyperski ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b00364 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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Enzyme engineering based on X-ray structures and

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kinetic profiling of substrate libraries: alcohol

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dehydrogenases for stereospecific synthesis of a

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broad range of chiral alcohols

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Yao Nie,†# Shanshan Wang,†‡# Yan Xu,†§* Shenggan Luo,¶ Yi-Lei Zhao,¶* Rong Xiao,∥⫫ Gaetano

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T. Montelione,∥ John F. Hunt,± and Thomas Szyperski⊥

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†

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Jiangnan University, Wuxi 214122, China.

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‡

School of Biotechnology, Key laboratory of Industrial Biotechnology, Ministry of Education,

School of Biological Science and Engineering, Shannxi University of Technology, Hanzhong

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723001, China.

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§

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China.

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¶

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Metabolic and Developmental Sciences, MOE-LSB & MOE-LSC, School of Life Sciences and

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Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

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∥Center

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Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA

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±

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122,

State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of

for Advanced Biotechnology and Medicine, Department of Molecular Biology and

Department of Biological Sciences, Columbia University, New York, NY 10027, USA

ACS Paragon Plus Environment

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⊥

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USA

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Department of Chemistry, The State University of New York at Buffalo, Buffalo, NY 14260,

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*E-mail: [email protected] (Y. Xu); [email protected] (Y.-L. Zhao)

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ABSTRACT: The narrow substrate scope of naturally occurring alcohol dehydrogenases

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(ADHs) greatly limits the enzymatic synthesis of important chiral alcohols. Based on X-ray

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crystal structures and kinetic profiling of a substrate library, we engineered variants of the

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stereospecific alcohol dehydrogenase from Candida parapsilopsis. This resulted in a set of four

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mutant enzymes which enable the asymmetric reduction of a broad range of prochiral ketones,

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including valuable pharmaceuticals and fine chemicals. The engineering strategy of this study

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paves the way for creating additional ADHs tailored for production of complex chiral alcohols.

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KEYWORDS: alcohol dehydrogenase, X-ray crystal structure, substrate library, kinetic

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profiling, stereoselectivity, substrate specificity

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INTRODUCTION

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Chiral alcohols are versatile building blocks for synthesis of pharmaceuticals and fine

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chemicals, such as antidepressants, anti-asthmatics, cholesterol-lowering agents, adrenergic

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receptor agonists, α- or β-adrenergic drugs, etc.1-3 For example, (R)-ethyl-4,4,4-trifluoro-3-

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hydroxybutanoate is the precursor for synthesizing befloxatone, a potent anti-depressant, and (S)-

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ethyl-3-hydroxy-3-phenylpropionate can be used to produce fluoxetine, tomoxetine, and

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nisoxetine.4,5 Alcohol dehydrogenases (ADH, EC 1.1.1.1-394) catalyze the reduction of prochiral

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ketones with very high chemo-, regio-, and stereoselectivitiy, and offer a valuable synthetic route

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for these alcohols.6-8 However, most naturally occurring ADHs exhibit a rather narrow substrate

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scope and thus do not reduce most of the ketone intermediates required for industrial

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applications.9

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Protein engineering provides approaches to expand the substrate scope of enzymes.10 In

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particular, structure-based redesign has been used to optimize the substrate scope for

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biotechnological important lipases,11,12 epoxide hydrolases13,14 and a large variety of

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oxidoreductases.15,16 In spite of an urgent demand, however, comparably few engineered ADHs

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have been reported so far.17-21

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Previously, we characterized the alcohol dehydrogenase/carbonyl reductase (EC 1.1.1.1)

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from Candida parapsilosis (CpRCR) as a robust, highly stereoselective biocatalyst following

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Prelog’s rule for ketone reduction,22-24 and determined the X-ray crystal structures in complex

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with ligands.25 However, CpRCR likewise exhibits a rather narrow substrate scope.26

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Here, we present the engineering of a set of CpRCR variants enabling stereospecific

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synthesis of broad range of chiral alcohols. We selected a representative substrate library


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containing 12 aryl ketones, 8 β-ketoesters, and 4 aliphatic ketones (Scheme 1), and measured the

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Michaelis-Menten kinetic parameters27 for wild-type (wt) CpRCR in order to accurately profile

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substrate specificity. Furthermore, we measured the enantiomeric excess (e.e.) for the products to

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profile stereoselectivity. Next, based on crystal structures and the profiling, we engineered

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CpRCR variants by replacing bulky residues forming the active site. The resulting catalytic

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efficiency and stereoselectivity was likewise profiled.

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Scheme 1.Substrate library consisting of ketones (1a-24a), where substituent RL is larger than

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RS. The CpRCR catalyzed reduction with NADH (shown at the top) yields the corresponding

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chiral alcohols (1b-24b), illustrated assuming that stereoselective reduction follows Prelog’s

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rule.22 The colors used for the substrates indicate how efficiently they are reduced by wt CpRCR

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(see Figure 1).

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RESULTS AND DISCUSSION

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Kinetic profiling of wt CpRCR towards the substrate library

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Figure 1. Catalytic profile of wt CpRCR towards 1a-24a of the substrate library (Scheme 1). A:

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Michaelis-Menten parameters. From left (21a) to right (19a), substrates are sorted by increasing

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molecular volume (for details see Table S1). For each substrate, Km (left axis), kcat (middle axis),

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and kcat/Km (right axis) are represented, respectively, by open, black and grey bars. Standard

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deviations for kcat and Km were obtained from triplicate measurements. B: Logarithmic graph

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(log2-scale) of kcat with the increase of molecular volume.

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Figure 1A provides the Michaelis-Menten kinetic parameters of wt CpRCR obtained for our

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substrate library (Scheme 1). The kcat value is considered to be the most important parameter for

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catalytic performance of enzymes28 under substrate saturation, as commonly encountered in

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biotechnological processes. Based on the kcat values, the substrates were thus divided into three

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classes (Figure 1B):

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Class (1): “good substrates” with high kcat > 4 s-1, located in the orange region of Figure 1B,

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includes the four substrates13a, 1a, 8a, and 14a. Compound 13a, the smallest β-ketoester, is

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most efficiently reduced by wt CpRCR exhibiting the highest kcat (8.98 s-1) along with good


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affinity (Km 0.25 mM), which results in high catalytic efficiency (kcat/Km 35.92 mM-1 s-1).

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Molecule 14a with a chlorinated methyl group as RS performs comparably. Substrates 1a and 8a,

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aryl ketones, exhibit kcat > 7 s-1 and good affinities (Km 0.50 mM and Km 0.95 mM, respectively).

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Hence, the substrates of class (1) are reduced very efficiently by wt CpRCR.

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Class (2): “moderate substrates” with kcat in the range 2-4 s-1, located in the yellow region of

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Figure 1B, includes 8 of the substrates. Catalytic activities are reduced by varying degrees

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towards the 1a-derivatives 2a, 6a, 7a, and 9a bearing substituents on the benzene ring (‘RL‘) and

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at the methyl group (‘RS‘). β-ketoester 17a with a phenyl group as RL exhibits good affinity (Km

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0.18 mM) and high catalytic efficiency (kcat/Km 14.28 mM-1s-1). Unexpectedly, for aliphatic

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ketones (21a, 23a, 24a), catalytic efficiency increases gradually along with the increase of the

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length of carbon chain of RL. Substrate 24a bearing hexyl group is more suitable for binding to

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the active site of wt CpRCR, as reflected by a lower Km (0.34 mM) and higher kcat (3.45 s-1) when

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compared with the propyl, butyl, and pentyl congeners.

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Class (3): “poor substrates” with kcat < 2 s-1, located in the blue region of Figure 1B,

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includes half (12) of the substrates of our library (Scheme 1). Although 3a and 4a have the same

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substituent at different C-position compared to 2a, the kcat values of 3a and 4a are reduced by

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varying degrees. Moreover, molecule 5a bearing bulky bromo substituent has a very low kcat

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value (0.83 s-1). The substrates 10a-12a, which have bulky aryl ketones, and β-ketoesters such as

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trihalogenated compounds (15a and 16a) and ethylbenzoyl acetate derivatives (18a and 19a), are

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also poor substrates for reduction with wt CpRCR.

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The kinetic data (Figure 1A) effectively profile the substrate specificity of wt CpRCR.

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Importantly, log(kcat) values are moderately anti-correlated with the molecular volume of the


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substrate (Figure 1B). This finding suggests that steric hindrance affects catalytic efficiency

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significantly. Since wt CpRCR exhibits kcat values which can be considered to be sufficiently

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high for biotechnology applications only for the four class (1) substrates (Scheme 1 and Figure

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1), engineering of CpRCR variants is required to improve catalytic efficiency towards class (2)

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and (3) substrates.

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Redesign of substrate-binding cavities

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We recently described the high-resolution X-ray crystal structures25 of wt CpRCR bound to

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NAD+ at 2.15 Å resolution (PDB ID: 3WLE), CpRCR mutant H49A bound to class 1 substrate

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8a at 2.95 Å (PDB ID: 3WNQ), and wt CpRCR bound to its product (R)-8b at 2.30Å (PDB ID:

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3WLF). These structures validated our detailed understanding of the catalytic mechanism of

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ADHs.8 The active site contains both a large and a small hydrophobic cavity close to the

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catalytic center, as well as the NADH binding site. The small cavity is formed mainly by the

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bulky side chains of F285 and W286, together with a segment of NADH (Figure 2). We

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anticipated that these two aromatic residues play an important role in determining substrate

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specificity. In the large cavity, the bulky side chain of residue W116 exhibits a hydrophobic

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interaction with the phenyl ring of substrate 8a in 3WNQ. Furthermore, significant spatial

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rearrangements of the side chains of W116, F285 and W286, creating a more “closed”

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conformation, were observed upon substrate binding (Figure S1).

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Based on our kinetic profiling data (Figure 1) and high-resolution structural information

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(Figure 2), we hypothesized that replacement of W116 or F285/W286 by Ala may (i) increase

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the sizes of the small and large cavities, respectively, and (ii) provide increased conformational

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flexibility of the active site to allow binding of a broader range of ketone substrates. In turn, we


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anticipated that such engineered enzymes may be suitable for catalyzing the reduction of class (2)

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and (3) substrates with greatly improved efficiency.

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Figure 2. Substrate-binding cavities in CpRCR (PDB ID: 3WLE) and key residues selected for

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engineering. A: Surfaces of the large (magenta) and small cavity (yellow) of CpRCR. B: Stick

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representation of the side chains of W116 (large cavity; magenta), F285, W286 and a segment of

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NAD+ (small cavity; yellow).

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Comparison of catalytic performance of the variants with wt CpRCR

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To test our hypothesis, we prepared samples of CpRCR mutants W116A, F285A, W286A,

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and F285A/W286A. Measurements of Michaelis-Menten parameters (Figures S2-S5) revealed

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dramatic changes in substrate specificity profiles and kcat values relative to wt CpRCR (Figure 3).

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For all four class (1) substrates (orange region in Figure 3), the ratios of kcat values (mutant/wt

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CpRCR) were around or below 1.0 for all mutants. This finding validates our conclusion that the

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active site of wt CpRCR is well tailored for small class (1) substrates. In contrast, for all class (2)

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substrates except 21a and 24a (yellow region), at least one of the mutant enzymes exhibits higher

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catalytic efficiency than wt CpRCR. Remarkably, kcat values for W116A, F285A, W286A, and

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F285A/W286A are increased to 2.6, 1.6, 4.2, and 4.4-fold towards 2a, respectively. For all class


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(3) substrates except for 12a and 15a (blue region), there is likewise at least one mutant which

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exhibits higher catalytic efficiency than wt CpRCR, and for nine substrates (22a, 3a, 4a, 5a, 20a,

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11a, 16a, 18a, 19a) the increase of kcat for one of the mutants is at least ~2 or higher. Specifically,

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W116A shows 2.9 and 3.0-fold increases of kcat towards substrates 16a and 19a, F285A/W286A

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exhibits 2.8, 2.9, 2.6, and 3.0-fold increases of kcat towards 22a, 4a, 11a, and 18a, and F285A

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shows 5.5, 8.8, and 8.5-fold increases of kcat for 3a, 20a, and 18a, respectively.

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Figure 3. Ratios of kcat values of mutants (W116A, F285A, W286A, and F285A/W286A) and wt

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CpRCR towards substrates 1a-24a (Scheme 1) sorted by class (1) (orange), class (2) (yellow),

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and class (3) (blue) (Figure 1).

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Overall, for all but four of the 20 class (2) and (3) substrates (Figure 1), at least one of the

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engineered mutants exhibits enhanced catalytic efficiency under substrate saturation (Figure 3).

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The enhanced catalytic performance can likely be attributed to a reshaped active site of the

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mutants: while wt CpRCR is suitable only for the smaller substrates, our engineered mutants can


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accommodate also more bulky substituents and exhibit a kinetic substrate profile which neatly

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complements wt CpRCR (Figure 3).

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Comparison of stereoselectivity of the variants with wt CpRCR

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Since active site engineering may well affect stereoselectivity of ketone reduction and thus

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enantiopurity of the alcohols,25 we also measured the optical purity of chiral alcohols resulting

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from reduction of compounds constituting our substrate library (Scheme 1) by either wt or

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mutant CpRCRs (Figure 4). The products of wt CpRCR-mediated reduction of 1a-24a follow

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Prelog’s rule.22 Likewise, W116A exhibits Prelog stereopreference for all ketones, while

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providing for some of them even higher stereoselectivity than wt CpRCR. This finding indicates

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that the W116A mutation within the large cavity does not significantly affect stereoselectivity.

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On the contrary, the W286A and F285A/W286A mutations dramatically affect stereoselectivity

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(Figure 4). W286A exhibits for 14 ketones Prelog, and for 10 ketones anti-Prelog

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stereoselectivity, with the e.e. values rarely exceeding 75%. F285A/W286A exhibits Prelog

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specificity for only 6 ketones and anti-Prelog stereoselectivity for 18 ketones. Remarkably, for

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eight of the anti-Prelog ketones (1a, 14a, 24a, 20a, 12a, 11a, 16a, and 17a), the e.e. exceeds

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80%, so that chiral alcohols can be synthesized which cannot be efficiently obtained with most of

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known naturally occurring ADHs. These findings show that reshaping of the small cavity of

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CpRCR can alter stereoselectivity. Substrate 12a represents the most striking example of our

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library: wt and W116A CpRCR yield the Prelog product with 100%e.e., while F285A, W286A,

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and F285A/W286A yield the anti-Prelog product with 100%e.e.


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Figure 4. Optical purity (%e.e.) of products from asymmetric reduction of 1a-24a, sorted by

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increasing molecular volume (Table S1) from left to right, catalyzed by purified wt CpRCR and

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mutants (for assay details see Table S2). Positive values of e.e. are expected following Prelog’s

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rule, while negative values indicate an anti-Prelog stereopreference.

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Overall, the combination of structure-based protein engineering with kinetic profiling of a

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substrate library represents a powerful approach for creating a set of four ADH variants offering

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broad substrate specificity and distinct stereoselectivity. Importantly, the kinetic profiling

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enables experimental assessment of the impact of entropic effects and structural rearrangements

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which are difficult to capture with computational methods.

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Computational study on substrate specificity and stereoselectivity

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To rationalize experimentally observed kinetic parameters (Figures 1 and 3, Figures S2-S5)

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and stereoselectivity (Figure 4) we initiated a comprehensive computational modeling study.

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First, to assess the impact of the mutations on the substrate-binding pocket, we performed 100 ns

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molecular dynamics (MD) simulations for both the wt CpRCR-NADH complex and the mutant


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congeners. Pocket volume distribution analysis (Figure 5) revealed that: (i) W286A (small cavity

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mutation) exhibits an average pocket volume and distribution which is quite similar to the wild

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type; (ii) F285A (small cavity mutation) and F285A/W286A (two small cavity mutations) exhibit

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larger average pocket volumes as well as broader distributions; and (iii) W116A (large cavity

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mutation) exhibits a smaller average pocket volume and a narrower distribution. As expected,

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these findings indicate that the mutations result in significant conformational rearrangements

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coming along with a significant and varying degree of ‘collapse’ of the binding pocket, which

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results from the replacement of the aromatic sidechains with a methyl group. Moreover, the

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broadening of the volume distributions suggest that some of these mutations result in increased

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conformational flexibility, which may enable the accommodation of a broader range of

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substrates.

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Figure 5. Distributions for pocket volumes of wt CpRCR and the mutants W116A, F285A,

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W286A, and F285A/W286A derived from 100 ns MD simulations.

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Second, we implemented a quantum mechanics/molecular mechanics (QM/MM)29 protocol

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enabling us to eventually develop a rationale for substrate stereoselectivity (Figure 4). The


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protocol was used to calculate the pro-S and pro-R transition states (TSs) for substrate 20a in wt

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CpRCR which lead, respectively, to ‘Prelog’ and ‘anti-Prelog’ products. It was quite

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straightforward to determine that, due to limited space in the active site pocket, the pro-R TS

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could be modeled only by ‘flipping’ of the RS group by a rotation around the carbonyl-RL bond

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(Figure 6). The similar result corresponding to the pro-S and pro-R TSs for substrate 20a in wt

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CpRCR was also obtained to the double mutant F285A/W286A (Figures S6). Consistent with the

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experimentally observed Prelog stereospecificity (Figure 4), the energy of the pro-S TS was

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calculated to be about 11.3 kcal/mol lower than that of the pro-R. Notably, the ‘RS group flip’

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might represent a distinct mechanism to explain inversion of stereospecificity in ADHs and may

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guide future engineering studies. Our QM/MM protocol shall be applied for other substrates as

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well as mutant enzymes to shed new light on the stereospecificity of ADHs in general.

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Figure 6. The superposition of pro-S (yellow, expected according the Prelog’s rule) and pro-R

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(green) transition states of substrate 20a in wt CpRCR.

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CONCLUSIONS B

A Ratio of kcat

0.5 0.8 1 1.5

2

3

4

F285A/W286A F285A/W286A W116A F285A W286A F285A/W286A F285A F285A/W286A F285A/W286A F285A F285A/W286A F285A/W286A W116A W116A W116A F285A/W286A F285A F285A F286A F285A/W286A W116A F285A/W286A F285A W116A

21a 13a 1a 22a 8a 2a 3a 4a 6a 15a 23a 5a 9a 7a 14a 24a 20a 10a 12a 11a 16a 17a 18a 19a

232

5

W116A F285A W286A

F285A/ W286A

e.e. (%)

100 90 75 50 25 0 -25 -50 -75 -90 -100

21a 13a 1a 22a 8a 2a 3a 4a 6a 15a 23a 5a 9a 7a 14a 24a 20a 10a 12a 11a 16a 17a 18a 19a

W116A WT W116A WT WT W116A WT WT W116A W116A W116A WT WT W116A WT WT W116A WT WT WT W116A WT WT WT WT

W116A F285A W286A

F285A/W286A F285A/W286A F285A/W286A W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A F285A/W286A

F285A/ W286A

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Figure 7. Comparison of catalytic performance of wt CpRCR with four engineered CpRCR

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mutants. A: Ratio of mutant to wt CpRCR kcat values for each compound in the substrate library

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(Scheme 1). The mutant with the largest ratio is indicated along with the corresponding color

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code in the right-most column. B: Enantiomeric excess (e.e.) of wt CpRCR and the four mutants

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for each compound. Positive e.e. values indicate synthesis following the Prelog’s rule, while the

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negative values represent anti-Prelog synthesis (see Table S2 for details). The enzyme with the

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highest positive or the lowest negative e.e. value is indicated along with the corresponding color

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code in the right-most two columns.

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We successfully altered the substrate specificity of CpRCR to allow reduction of a

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broadened range of substrates, including significantly more bulky ketones (Figure 7). The

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resulting set of four CpRCR mutants is now available for biotechnological applications (notably,

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these four mutants were the only ones we expressed and tested). Furthermore, our strategy


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allowed us to create mutants which either retain or reverse stereoselectivity of wt CpRCR, and

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we initiated a computational study to rationalize our findings. In particular, the reversal of

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stereoselectivity observed for selected variants and substrates enables the synthesis of chiral

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alcohols which thus far can not be obtained with known naturally occurring ADHs. Figure 7

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exemplifies the high complementarity of wt CpRCR together with four specific mutants for

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synthesis of a broad range of alcohols with varying stereoselectivity. Depending on the desired

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substrate and stereoselectivity, this analysis enables one to choose the best CpRCR variant. It is

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evident that the engineering strategy described here can be employed to generate a suitably

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expanded set of CpRCR mutant proteins.

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EXPERIMENTAL SECTION

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Materials

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Enzymes, vectors, oligonucleotides and other reagents for DNA cloning and amplification

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were obtained from Takara-Bio Co., Japan and Novagen Co., USA. Substrates (1a-24a) were

259

purchased from Sigma-Aldrich (USA) or TCI (Shanghai). NADH, NAD+, (R)-/(S)-1a,(R)-/(S)-8a

260

were purchased from Sigma-Aldrich (USA). All other racemic alcohol standards were prepared

261

by reduction of ketones with sodium borohydride. Hexane and isopropanol used for High

262

Performance Liquid Chromatography (HPLC) were of chromatographic grade (Honeywell Co.,

263

USA). All other chemicals used were of analytical grade and commercially available.

264

Expression and purification of wt CpRCR and variants


15

ACS Catalysis 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 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 36

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Recombinant strains of wt CpRCR and the variants (F285A, W286A, and F285A/W286A)

266

were constructed previously using the pET32 Xa/LIC vector.25 The mutation W116A was

267

introduced by using the protocol of the Agilent QuikChange Lightning Site-Directed

268

Mutagenesis Kit. Importantly, the four mutants (W116A, F285A, W286A, and F285A/W286A)

269

were

270

GTAAAAACGCATTTGGTGATGCGTTCGGATTGGGGTACGATGGTG - 3’, W116A_R: 5’-

271

CACCATCGTACCCCAATCCGAACGCATCACCAAATGCGTTTTTAC - 3’. Plasmid DNA

272

was prepared and sequenced to validate positive clones. Recombinant plasmids were transformed

273

in Escherichia coli BL21 trxB (DE3). Cells were grown in LB medium containing 100 µg mL-1

274

ampicillin, 50 µg mL-1 kanamycin, and 0.2 mM zinc acetate. The protein was expressed without

275

induction of isopropyl β-D-1-thiogalactopyranoside (IPTG) at 28 °C for 12 hours.30 Cells were

276

harvested by centrifugation at 7,000 ×g and resuspended in the lysis buffer (pH 8.0) containing

277

25 mM Tris-HCl, 150 mM NaCl, and 20 mM imidazole. Cell lysates were prepared with a

278

French Press, and then centrifuged at 17,000 ×g to remove cell debris. Proteins were purified by

279

FPLC using a Ni-NTA column. The buffer and gradient for the Ni-NTA column were 25 mM

280

Tris-HCl, 150 mM NaCl, and 20-500 mM imidazole, pH 8.0. His-tagged CpRCR was eluted at

281

about 120 mM imidazole. The protein solution was dialyzed against the buffer containing 25

282

mM Tris-HCl, 150 mM NaCl, pH 8.0, and then subjected to Factor Xa digestion to remove the

283

His-tag. The mixture was then passed through Ni-NTA column again and the untagged CpRCR

284

was eluted with 5 mM imidazole-containing buffer. The purified protein was finally concentrated

285

to 3 mg mL-1 in the buffer (pH 8.0) containing 25 mM Tris-HCl and 150 mM NaCl.

286

Determination of kinetic parameters

the

only

ones

constructed

for

our

study.

Primers

were:

W116A_F:

5’-


16

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ACS Catalysis

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The standard assay mixture in 100 µL for the enzyme activity of the purified wt CpRCR and

288

the variants comprised 0.1 M potassium phosphate buffer (pH 6.5), 5 mM ZnCl2, 1 mM NADH,

289

5 mM substrates, and an appropriate amount of enzyme. The decrease in NADH absorbance at

290

340 nm was recorded for 3 min at 30 °C. A molar extinction coefficient of 6,220 L mol-1 cm-1

291

was used for NADH. One unit (U) of enzyme activity was defined as 1 µmol of NADH

292

consumed per minute under the assay conditions. Kinetic parameters of wt CpRCR and the

293

variants towards various substrates were assayed by measuring initial velocity at various

294

concentrations of substrates and cofactor.25 To determine the apparent Km value, the

295

concentration of carbonyl compounds was varied from 0.1 to 8 mM with a fixed concentration of

296

NADH at 0.05 mM, 0.1 mM, and 0.25 mM, respectively. The reaction follows a Theorell-

297

Chance BiBi mechanism.27 The Michaelis-Menten kinetic parameters were obtained from a non-

298

linear least-squres fit of the Michaelis-Menten equation to the experimental data and further

299

validated from double reciprocal Lineweaver–Burk plots. All the data were averaged from three

300

replicates for each substrate and cofactor concentration and significant differences (p

Enzyme Engineering Based on X-ray Structures and Kinetic Profiling

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