The X‐Ray Crystal Structure of a Mutant Glycerol Dehydrogenase from Bacillus coagulans with Acquired Lactate Dehydrogenase Activity

Abstract

Successful metabolic evolution of an industrial biocatalyst depends on accumulation of desired genetic changes that enhance product titer, yield and productivity. During the development of a strain of Bacillus coagulans for production of D‐lactic acid, a glycerol dehydrogenase (GDH) evolved to catalyze reduction of pyruvic acid to D‐lactic acid (D‐LDH) by acquisition of two mutations, D121N and F245S. The native GDH oxidized glycerol and a variety of diols, but displayed very low D‐LDH activity. The evolved enzyme has a 10‐fold lower level of glycerol oxidation activity and a 15‐fold higher D‐LDH activity than the wild type enzyme. In order to explain these kinetic data, we have determined the x‐ray crystallographic structure of B. coagulans GDH D121N/F245S. The protein crystallized within 48 hours in 0.2M ammonium citrate dibasic, pH 4.5 with 10% PEG3350 at room temperature. The crystals belonged to the orthorhombic space group C2221 and had unit cell parameters of 211 Å, 211 Å and 149 Å with α=β=γ=90°. Diffraction data were collected to 2.5 Å resolution on beamline 22‐ID at the Advanced Photon Source at Argonne National Laboratory, and an initial model was created using the automated molecular replacement software MrBUMP. Multiple rounds of model building and refinement were performed using Coot and Refmac, respectively. Comparison of the mutant protein to a homologous protein from Geobacillus stearothermophilus has revealed that the acquired mutations in the B. coagulans GDH D121N/F245S protein significantly alter the geometry of the active site. The active site of the double mutant enzyme is better able to bind carboxyl functionalities in substrates in addition to having a decreased affinity for the hydroxyl groups of the original substrate diols.