Expression, Purification, Crystallization and Preliminary X-ray Crystallographic Analysis of L-lactate Dehydrogenase and Its H171C Mutant from Bacillus Subtilis

Overview

Journal Acta Crystallogr Sect F Struct Biol Cryst Commun

Date 2012 Jan 11

PMID 22232174

Citations 2

Authors

Yanfeng Zhang

Xiaoli Gao

Affiliations

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Abstract

L-Lactate dehydrogenase (LDH) is an important enzyme involved in the last step of glycolysis that catalyzes the reversible conversion of pyruvate to L-lactate with the simultaneous oxidation of NADH to NAD(+). In this study, wild-type LDH from Bacillus subtilis (BsLDH-WT) and the H171C mutant (BsLDH-H171C) were expressed in Escherichia coli and purified to near-homogeneity. BsLDH-WT was crystallized in the presence of fructose 1,6-bisphosphate (FBP) and NAD(+) and the crystal diffracted to 2.38 Å resolution. The crystal belonged to space group P3, with unit-cell parameters a = b = 171.04, c = 96.27 Å. BsLDH-H171C was also crystallized as the apoenzyme and in complex with NAD(+), and data sets were collected to 2.20 and 2.49 Å resolution, respectively. Both BsLDH-H171C crystals belonged to space group P3, with unit-cell parameters a = b = 133.41, c = 99.34 Å and a = b = 133.43, c = 99.09 Å, respectively. Tetramers were observed in the asymmetric units of all three crystals.

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