Expression, purification, crystallization and preliminary X-ray crystallographic analysis of L-lactate dehydrogenase and its H171C mutant from Bacillus subtilis.

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 A resolution. The crystal belonged to space group P3, with unit-cell parameters a = b = 171.04, c = 96.27 A. BsLDH-H171C was also crystallized as the apoenzyme and in complex with NAD+, and data sets were collected to 2.20 and 2.49 A resolution, respectively. Both BsLDH-H171C crystals belonged to space group P3, with unit-cell parameters a = b = 133.41, c = 99.34 A and a = b = 133.43, c = 99.09 A, respectively. Tetramers were observed in the asymmetric units of all three crystals.