Gene mining, codon optimization and analysis of binding mechanism of an aldo-keto reductase with high activity, better substrate specificity and excellent solvent tolerance.

Vinod Kumar Vashistha,Weiliang Wu,Wei Jiang,Xiaoli Fu

doi:10.1371/journal.pone.0260787

Abstract

The biosynthesis of chiral alcohols has important value and high attention. Aldo–keto reductases (AKRs) mediated reduction of prochiral carbonyl compounds is an interesting way of synthesizing single enantiomers of chiral alcohols due to the high enantio-, chemo- and regioselectivity of the enzymes. However, relatively little research has been done on characterization and apply of AKRs to asymmetric synthesis of chiral alcohols. In this study, the AKR from Candida tropicalis MYA-3404 (C. tropicalis MYA-3404), was mined and characterized. The AKR shown wider optimum temperature and pH. The AKR exhibited varying degrees of catalytic activity for different substrates, suggesting that the AKR can catalyze a variety of substrates. It is worth mentioning that the AKR could catalytic reduction of keto compounds with benzene rings, such as cetophenone and phenoxyacetone. The AKR exhibited activity on N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP), a key intermediate for biosynthesis of the antidepressant drug duloxetine. Besides, the AKR still has high activity whether in a reaction system containing 10%-30% V/V organic solvent. What’s more, the AKR showed the strongest stability in six common organic solvents, DMSO, acetonitrile, ethyl acetate, isopropanol, ethanol, and methanol. And, it retains more that 70% enzyme activity after 6 hours, suggesting that the AKR has strong solvent tolerance. Furthermore, the protein sequences of the AKR and its homology were compared, and a 3D model of the AKR docking with coenzyme NADPH were constructed. And the important catalytic and binding sites were identified to explore the binding mechanism of the enzyme and its coenzyme. These properties, predominant organic solvents resistance and extensive substrate spectrum, of the AKR making it has potential applications in the pharmaceutical field.

Highlights

The living example of the synthesis of chiral alcohols by aldo-keto reductases (AKRs) are few compared to alcohol dehydrogenase and carbonyl reductase, and, it was deserved to be studied [25, 26]
It is worth mentioning that the AKR could catalytic reduction of keto compounds with benzene rings, such as acetophenone and phenoxyacetone
The AKR exhibited about 20% relative activity on DKTP, the key intermediate for biosynthesis of the antidepressant drug duloxetine [27, 29]. These results indicate that the AKR could be employed to synthesis of chiral alcohol compounds containing a benzene ring and duloxetine, so the enzyme has a high application potential

Summary

Introduction

Biocatalysis has shown advantages and concerns due to its high chiral selectivity, stereoscopic and regional selectivity, environmental friendliness, mild conditions and so on. Enzymes, such as alcohol dehydrogenase, ketone reductase, etc, are the key to the synthesis of chiral compounds by biocatalysis [7,8,9,10,11,12,13,14,15]. The aldo-keto reductases (AKRs) could catalyze the reduction of ketones or different acids to chiral alcohols [16,17,18]. The living example of the synthesis of chiral alcohols by AKRs are few compared to alcohol dehydrogenase and carbonyl reductase, and, it was deserved to be studied [25, 26]

Methods

Findings

Discussion

Conclusion