Modified catalytic performance of Lactobacillus fermentum l-lactate dehydrogenase by rational design

Aiai Wu,Yujie Cai,Xiaohui Zheng,Yajun Bai,Taiping Fan

doi:10.1007/s43393-021-00067-3

Abstract

l-Lactate dehydrogenases can reduce alpha-keto carboxylic acids asymmetrically and generally have a broad substrate spectrum. l-Lactate dehydrogenase gene (LF-l-LDH0845) with reducing activity towards 3,4-dihydroxyphenylpyruvate and phenylpyruvate was obtained from Lactobacillus fermentum JN248. To change the substrate specificity of LDH0845 and improve its catalytic activity towards large substrates, site-directed mutation of Tyr221 was performed by analyzing the amino acids in the active center. Kinetic parameters show that the kcat values of Y221F mutant on 3,4-dihydroxyphenylpyruvate, 4-methyl-2-oxopentanoate, and glyoxylate are 1.21 s−1, 1.35 s−1, and 0.72 s−1, respectively, which are 420%, 150% and 130% of the wild-type LDH0845. This study shows that the mutations of Y221 can significantly change the substrate specificity of LDH0845, making it become a potential tool enzyme for the reduction of alpha-keto carboxylic acids with large functional groups.

Highlights

Introduction lLactate dehydrogenase (l-LDH, EC 1.1.1.27), an oxidoreductase of N AD+ dependent l-specific hydroxy acid dehydrogenases family, exists in human beings, animals, plants and microorganisms in the form of tetramer (LDHM4, LDH-M3H, LDH-M2H2, LDH-M3H, LDH-H4) [7]
After the modeling result is verified (Supplementary Note 2), NADH and natural substrate pyruvate were docked with LDH0845
Ile224 is close to the side chain of the substrate, occupying the space of the active pocket, which may affect the substrate specificity of the enzyme

Summary

Introduction

Introduction lLactate dehydrogenase (l-LDH, EC 1.1.1.27), an oxidoreductase of N AD+ dependent l-specific hydroxy acid dehydrogenases family, exists in human beings, animals, plants and microorganisms in the form of tetramer (LDHM4, LDH-M3H, LDH-M2H2, LDH-M3H, LDH-H4) [7]. LDH can catalyze pyruvate to lactic acid. When the animal lacks glucose, LDH can oxidize lactic acid to pyruvate, which is converted into glucose through gluconeogenesis. LDH can be used as a biomarker for cancer diagnosis and prognosis. LDH is considered as a promising target for the prevention and treatment of cancer [12]. LDH is used to produce lactic acid and phenyllactic acid [23] and can produce glycolic, mandelic acid and 3,4-dihydroxyphenyllactic acid. Those different alpha-hydroxy acids obtained were widely used in industrial production [39, 42, 45, 48]

Methods

Results

Discussion

Conclusion