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 (Km = 0.04 mM) and NADPH (Km = 0.06 mM) as cofactors, uses hydroxypyruvate (Km = 0.1 mM) and glyoxylate (Km = 1.5 mM) as the only substrates for the forward reaction, and carries out the reverse reaction with glycerate (Km = 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.Methylobacterium extorquens AM1 is a gram-negative facultative methylotroph capable of growth on methanol, methylamine, and a variety of multicarbon compounds which assimilates C1 units via the serine cycle during growth on C1 substrates (12,17). A number of enzymes have been shown to be specifically involved in dissimilation and assimilation of C1 substrates in M. extorquens AM1, and these are known to be inducible by growth on the appropriate C1 substrates. In addition, the assimilatory enzymes are repressed by the presence of multicarbon compounds such as succinate or glucose, but the dissimilatory enzymes are not (2,14).Although intensive work on the methanol oxidation system in M. extorquens AM1 is beginning to shed light on both the biochemistry and the regulatory mechanisms involved (1,13,15,16,18), the serine cycle is less well understood. This cycle functions by the condensation of methylene tetrahydrofolate and glycine to generate serine, conversion of serine to phosphoenolpyruvate via several steps, and carboxylation of phosphoenolpyruvate to generate oxaloacetate. The oxaloacetate is then converted to malyl coenzyme A (CoA), which is cleaved to acetyl-CoA and glyoxylate. The acetyl-CoA is oxidized to glyoxylate, and this is then converted to the acceptor molecule, glycine. However, the route of acetyl-CoA oxidation in this last stage is unknown. Isocitrate lyase is not present in this organism, and a novel, as yet undetermined pathway must carry out this oxidation (2).Hydroxypyruvate reductase catalyzes the reduction of hydroxypyruvate to glycerate, one of the key steps in the conversion of serine to phosphoenolpyruvate in the serine cycle. It has also been suggested to carry out the reduction of glyoxylate to glycolate in cell extracts, but the significance of this reaction in vivo is not known (2, 4). Mutants in hydroxypyruvate reductase would be expected to be defective in growth on C1 compounds, but should grow normally on other compounds. However, mutant 20BL, which lacks hydroxypyruvate reductase, is unable to grow on both C1 and C2 compounds (4). This suggests a second role for * Corresponding author.hydroxypyruvate reductase involving C2 compounds, most likely, the oxidation of glycolate to glyoxylate (4...