Specific coenzyme preference switching for an aldo-keto reductase that synthesizes the chiral intermediate of duloxetine

Lingzhi Zhang; Rui Zhou; Xiaoli Fu; Guangya Zhang; Lijuan Zhang; Shu-Feng Zhou; Wei Jiang

doi:10.1016/j.enzmictec.2023.110326

Specific coenzyme preference switching for an aldo-keto reductase that synthesizes the chiral intermediate of duloxetine

Enzyme Microb Technol. 2023 Dec:171:110326. doi: 10.1016/j.enzmictec.2023.110326. Epub 2023 Sep 13.

Authors

Lingzhi Zhang¹, Rui Zhou¹, Xiaoli Fu¹, Guangya Zhang¹, Lijuan Zhang², Shu-Feng Zhou³, Wei Jiang⁴

Affiliations

¹ College of Chemical Engineering, Huaqiao University, Xiamen 361021, Fujian Province, PR China.
² State Key Laboratory of Microbial Technology, Shandong University, No. 72 Binhai Road, Qingdao 266237, PR China.
³ College of Chemical Engineering, Huaqiao University, Xiamen 361021, Fujian Province, PR China. Electronic address: szhou@hqu.edu.cn.
⁴ College of Chemical Engineering, Huaqiao University, Xiamen 361021, Fujian Province, PR China. Electronic address: wjiang@hqu.edu.cn.

PMID: 37717530
DOI: 10.1016/j.enzmictec.2023.110326

Abstract

The synthesis of chiral intermediates for the traditional antidepressant duloxetine has gained significant attention as the number of depressed patients continues to grow. S-N, N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamide (S-DHTP) is a critical intermediate in the synthesis of duloxetine, and the chemical synthesis process is complex and environmentally unfriendly. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a major cost driver in the biocatalytic production of S-DHTP from N, N-Dimethyl-3-keto-3-(2-thienyl)-1-propanamide (DKTP). Here, we successfully modified the coenzyme preference of an aldo-keto reductase (AKR7-2-1) to use the cheaper reduced nicotinamide adenine dinucleotide (NADH) through a coenzyme preference modification approach. We utilized protein engineering to create a superior mutant, Y53F, which increased the coenzyme specificity of AKR7-2-1 by 875-fold and improved its thermal stability, enhancing its potential for industrial applications. Molecular dynamics simulations were performed to demonstrate the effect of mutations at key sites on the protein, revealing the altered coenzyme preference and increased thermal stability from structural and energetic changes. This study validates the viability of the coenzyme preference modification strategy for aldo-keto reductase, offering valuable insights for fellow researchers and guiding future investigations.

Keywords: Aldo-keto reductase; Biocatalysis; Duloxetine; Molecular docking; Molecular dynamics simulation; Reversal of cofactor dependence.