Characterization of the Pro101Gln mutation that enhances the catalytic performance of T. indicus NADH-dependent d-lactate dehydrogenase

Jiongqin Liu; Gongquan Liu; Xiao Han; Fei Tao; Ping Xu

doi:10.1016/j.str.2023.08.019

Characterization of the Pro101Gln mutation that enhances the catalytic performance of T. indicus NADH-dependent d-lactate dehydrogenase

Structure. 2023 Dec 7;31(12):1616-1628.e3. doi: 10.1016/j.str.2023.08.019. Epub 2023 Sep 19.

Authors

Jiongqin Liu¹, Gongquan Liu¹, Xiao Han¹, Fei Tao², Ping Xu³

Affiliations

¹ State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
² State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China. Electronic address: taofei@sjtu.edu.cn.
³ State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China. Electronic address: pingxu@sjtu.edu.cn.

PMID: 37729918
DOI: 10.1016/j.str.2023.08.019

Abstract

NADH-dependent d-lactate dehydrogenases (d-LDH) are important for the industrial production of d-lactic acid. Here, we identify and characterize an improved d-lactate dehydrogenase mutant (d-LDH1) that contains the Pro101Gln mutation. The specific enzyme activities of d-LDH1 toward pyruvate and NADH are 21.8- and 11.0-fold greater compared to the wild-type enzyme. We determined the crystal structure of Apo-d-LDH1 at 2.65 Å resolution. Based on our structural analysis and docking studies, we explain the differences in activity with an altered binding conformation of NADH in d-LDH1. The role of the conserved residue Pro101 in d-LDH was further probed in site-directed mutagenesis experiments. We introduced d-LDH1 into Bacillus licheniformis yielding a d-lactic acid production of 145.9 g L^-1 within 60 h at 50°C, which was three times higher than that of the wild-type enzyme. The discovery of d-LDH1 will pave the way for the efficient production of d-lactic acid by thermophilic bacteria.

Keywords: NADH-dependent dehydrogenase; adaptive evolution; binding conformation; catalytic performance; d-lactate dehydrogenase; d-lactic acid.

MeSH terms

L-Lactate Dehydrogenase* / chemistry
L-Lactate Dehydrogenase* / genetics
L-Lactate Dehydrogenase* / metabolism
Lactic Acid / chemistry
Lactic Acid / metabolism
Mutation
NAD* / chemistry

Substances

L-Lactate Dehydrogenase
D-lactate dehydrogenase
NAD
Lactic Acid