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 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.
Genetics Graduate Program, Michigan State University, East Lansing, MI 48824, USA. zhangy11@msu.edu
Yanfeng
Y
Zhang
Xiaoli
X
Gao
D001412:Bacillus subtilis / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation
D007770:L-Lactate Dehydrogenase