Structure-guided engineering of ChKRED20 from Chryseobacterium sp. CA49 for asymmetric reduction of aryl ketoesters

https://doi.org/10.1016/j.enzmictec.2019.03.001Get rights and content

Highlights

  • The X-ray crystal structure of ChKRED20/NAD+ complex was solved.

  • Three rounds of iterative saturation mutagenesis were performed.

  • Mutants with increased substrate acceptance toward aryl ketoesters were achieved.

  • One mutant had a kcat/Km 196-fold of the wild-type.

  • All three substrates were completely reduced at 100 g/l loading.

Abstract

ChKRED20 is a robust NADH-dependent ketoreductase identified from the genome of Chryseobacterium sp. CA49 that can use 2-propanol as the ultimate reducing agent. The wild-type can reduce over 100 g/l ketones for some pharmaceutical relevant substrates, exhibiting a remarkable potential for industrial application. In this work, to overcome the limitation of ChKRED20 to aryl ketoesters, we first refined the X-ray crystal structure of ChKRED20/NAD+ complex at a resolution of 1.6 Å, and then performed three rounds of iterative saturation mutagenesis at critical amino acid sites to reshape the active cavity of the enzyme. For methyl 2-oxo-2-phenylacetate and ethyl 3-oxo-3-phenylpropanoate, several gain-of-activity mutants were achieved, and for ethyl 2-oxo-4-phenylbutanoate, improved mutants were achieved with kcat/Km increasing to 196-fold of the wild-type. All three substrates were completely reduced at 100 g/l loading catalyzed with selected ChKRED20 mutants, and deliver the corresponding chiral alcohols with >90% isolated yield and 97 - >99%ee.

Introduction

Stereoselective ketone reduction is a dominant approach for the production of chiral alcohols in organic synthesis. Ketoreductases (KREDs) are known to catalyze such a reaction in the presence of NAD(P)H with high efficiency and selectivity under mild conditions [[1], [2], [3], [4], [5],[6], [7], [8], [9]]. A variety of KREDs have been successfully applied in the industrial-scale production of many pharmaceutical intermediates, such as those of atorvastatin, duloxetine, atazanavir, crizotinib and ezetimibe [[10], [11], [12], [13], [14], [15]]. For many biocatalytic processes, protein engineering has proven to be a powerful and crucial tool that enables the development of mutant enzymes that overcome the limitations of the wild-type enzyme, such as narrow substrate scope, low catalytic activity, or insufficient tolerance toward temperature and organic solvent [[16], [17], [18], [19], [20], [21], [22]].

ChKRED20 is an anti-Prelog short-chain reductase/dehydrogenase (SDR) identified and characterized by our research group from the genome of Chryseobacterium sp. CA49 (GenBank accession No. KC342020). It catalyzes the stereoselective reduction of a spectrum of ketones to the corresponding chiral alcohols, including the key intermediates for the synthesis of aprepitant, ticagrelor and atorvastatin [[23], [24], [25], [26], [27]]. The wild-type ChKRED20 is a remarkably efficient enzyme that often allows the complete conversion of 100–300 g/l substrate within hours. Another advantage of this enzyme is the ability to use 2-propanol as the ultimate reducing agent to recycle NADH, and the high tolerance to 2-propanol, which makes it very attractive for industrial applications [24].

Recently, we have solved the X-ray crystal structure of the apo-enzyme of ChKRED20, and subsequently, expanded the catalytic scope to ortho-substituted acetophenones through iterative saturation mutagenesis [28]. In order to further exploit the potential of ChKRED20 for the production of diversified chiral alcohols with excellent activity and stereoselectivity, the present work focuses on aryl ketoesters, another group of important precursors. The corresponding products are chiral hydroxy esters, which severe as important building blocks for the synthesis of many pharmaceuticals and fine chemicals [2,29,30]. We have reported that the wild-type ChKRED20 and mutants can catalyze the reduction of ethyl 4-chloro-3-oxobutanoate, an aliphatic ketoester, at 300 g/l with a space-time yield of 1824 mM/h, delivering the chiral intermediate for the synthesis of “blockbuster” drug statins [24]. However, aryl ketoesters are not well accepted as substrates for the wild-type ChKRED20. Typical aryl α-ketoester and β-ketoester, such as methyl 2-oxo-2-phenylacetate (1a) and ethyl 3-oxo-3-phenylpropanoate (2a) remain unchanged in a catalysis system based on ChKRED20.

In the current work, to develop mutants for the reduction of aryl ketoesters, we intend to first solve the X-ray crystal structure of the ChKRED20/NAD+ complex, in hope of a more reliable prediction of critical amino acid residues than using the structure of the cofactor-free apo-enzyme as in our previous work [28]. The resulting structure of the complex guided three rounds of double- or single-sited saturation mutagenesis, which resulted in several mutants that were suitable for the biocatalytic reduction of two α-ketoesters and one β-ketoester at a substrate loading of 100 g/l.

Section snippets

Materials

Substrates methyl 2-oxo-2-phenylacetate (1a), ethyl 3-oxo-3-phenylpropanoate (2a), and ethyl 2-oxo-4-phenylbutanoate (3a) were purchased from Alfa-Aesar (Tianjin, China). Product standards, (1R)- and (1S)-methyl 2-hydroxy-2-phenylacetate (1b), (rac)-ethyl 3-hydroxy-3-phenylpropanoate (2b) and (2R)- and (2S)-ethyl 3-hydroxy-3-phenylpropanoate (3b) were purchased from J&K Scientific Ltd. (Shanghai). Enzymes used for DNA manipulation were purchased from New England Biolabs (Beverly, MA, USA).

X-ray crystal structure of ChKRED20/NAD+ complex

The crystal structure of the ChKRED20/NAD+ complex was refined at 1.60 Å resolution (PDB ID: 6IXM), exhibiting a homotetrameric quaternary structure (Fig. 2). In three out of four subunits of the asymmetric unit were observed electron density corresponding to the cofactor NAD+, which was situated in a crevice at the center of the Rossmann-fold. The Cα atoms between the apo-ChKRED20 without any cofactor (PDB code: 5 × 8H) [28] and the ChKRED20/NAD+ complex fit within an RMSD of 0.4 Å with a

Conclusions

We have solved the crystal structure of the ChKRED20/NAD+ complex at 1.6 Å resolution, successfully identified six key residues surrounding the substrate-binding pocket in ChKRED20 that affect the catalytic activity, and subsequently developed several mutants with significantly increased substrate acceptance toward aryl ketoesters. The availability of the crystal structures of both apo-ChKRED20 and ChKRED20/NAD+ complex may provide great advantage for further exploitation of this

Acknowledgments

Shanghai Synchrotron Radiation Facilities (SSRF) and National Center for Protein Science Shanghai (NCPSS), China are gratefully acknowledged for the provision of synchrotron radiation facilities and efficient support. This work was supported by the National Natural Science Foundation of China21708038 (to Y. Liu) and 21572220 (to Z.-L. Wu), and the Open Fund of Key Laboratory of Environmental and Applied Microbiology KLCAS-2016-08 (to Y. Liu & Z.-G. Zhang) of the Chinese Academy of Sciences.

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