Studies on the Metabolic Enzymes Characteristic to C_1-Microorganisms----Structures and Functions

点击翻译按钮获取中文摘要

The serine pathway is a characteristic microbial assimilation pathway of C_1 compounds such as methanol. The pathway includes methanol dehydrogenase (MDH) and serine hydroxymethyltransferase (SHMT), by both of which C_1 compounds are elongated to C_3 compounds, L-serine. Thus, one can expect a conversion rate of 100% of glycine to L-serine with methanol supply. The present studies revealed the enzymatic, protein-chemical, molecular genetical, and crystallographic elucidation of the enzymes related to the serine pathway, especially hydroxypyruvate reductase (HPR)), of the serine-producing methanol-utilizing bacterium, Hyphomicrobium methylovorum.HPR of strain GM2 was purified to homogeneity and crystallized, the first time for an enzyme from a methylotroph. The enzyme was found to be a dimer composed of identical subunits (38 kDa), the molecular mass of the enzyme being about 70 kDa. The enzyme was stable against heating at 25 C for 10 min at pH values beween 5 and 9. Optimal activity w … More as observed at pH 6.8 and around 45^-C.The enzyme catalyzed the reduction of hydroxypyruvate with the oxidation of only NADH.Other than hydroxypyruvate, only glyoxylate served as a substrate. The Km values were found to 0.175 mM for hydroxypyruvate and 10.8mM for glyoxylate. X-ray crystallographic studies of the HPR revealed that the enzyme molecule is a symmetrical dimer composed of subunits of molecular mass 38kDa, and shares significant structural homology with another NAD-dependent enzyme, formate dehydrogenase. The HPR subunit consistes of two structurally similar domains that are approximately related to each other by 2-fold symmetry. The domains are separated by a deep cleft that forms the purtative NAD and substrate binding sites. One of the domains has been identified as the NAD-binding domain based on its close structural similarity ot the NAD-binding domains of other NAD-dependent dehydrogenases. The topology of the second domain is different from that found in the various catalytic domains of other dehydrogenases. This is the first report of the crystallographic study of D-specific 2-hydroxy acid dehydrogenase. Less