Cloning, expression, purification, crystallization and X-ray crystallographic analysis of d-lactate dehydrogenase from Lactobacillus jensenii

Abstract

The thermostable d-lactate dehydrogenase from Lactobacillus jensenii (Lj d-LDH) is a key enzyme for the production of the d-form of lactic acid from pyruvate concomitant with the oxidation of NADH to NAD+. The polymers of lactic acid are used as biodegradable bioplastics. The Lj d-LDH protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 28%(w/v) polyethylene glycol 400, 100 mM Tris-HCl pH 9, 200 mM magnesium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 A. The crystal belonged to space group P3121, with unit-cell parameters a = b = 90.5, c = 157.8 A. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.58 A3 Da-1, which corresponds to a solvent content of approximately 52.3%. The structure was solved by single-wavelength anomalous dispersion using a selenomethionine derivative.