Preparation of Recombinant Formate Dehydrogenase from Thermotolerant Yeast Ogataea parapolymorpha and Crystallization of Its Apo- and Holo- Forms

Abstract

NAD+-dependent formate dehydrogenase from thermotolerant yeast Ogataea parapolymorpha DL-1 (OpaFDH, EC 1.2.1.2) with the additional N-terminal Gly residue and its double mutant OpaFDH_AD were overexpressed in E. coli cells. The enzyme yield was, respectively, 6000 and 6200 units per liter of the cultivation medium. Purified enzymes were obtained as homogeneous preparations with a yield of 62%. The purification procedure included ultrasonic cell disruption, heat treatment of cell free extracts at 55°C for 15 min, and hydrophobic chromatography on the Phenyl Sepharose Fast Flow. Crystallization experiments with wild-type OpaFDH resulted in the preparation of crystals of the apo-form but not the holo-form. Crystals of the holo-form were prepared in the case of the OpaFDH_AD mutant in the presence of 7 mM NAD+ and 10 mM sodium azide. The size and quality of the crystals are sufficient to collect X-ray diffraction data and determine the enzyme three-dimensional structure.