Purification and Characterization of Hydroxypyruvate Reductase from the Facultative Methylotroph Methylobacterium extorquens AM1

Abstract

Hydroxypyruvate reductase was purified to homogeneity from the facultative methylotroph Methylobacterium extorquens AM1. It has a molecular mass of about 71 kDa, and it consists of two identical subunits with a molecular mass of about 37 kDa. This enzyme uses both NADH (K_m = 0.04 mM) and NADPH (K_m = 0.06 mM) as cofactors, uses hydroxypyruvate (K_m = 0.1 mM) and glyoxylate (K_m = 1.5 mM) as the only substrates for the forward reaction, and carries out the reverse reaction with glycerate (K_m = 2.6 mM) only. It was not possible to detect the conversion of glycolate to glyoxylate, a proposed role for this enzyme. Kinetics and inhibitory studies of the enzyme from M. extorquens AM1 suggest that hydroxypyruvate reductase is not a site for regulation of the serine cycle at the level of enzyme activity.