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

C_1-微生物代谢酶特性的研究----结构与功能

基本信息

批准号：
06044148
负责人：
IZUMI Yoshikazu
金额：
$ 3.14万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1994
资助国家：
日本
起止时间：
1994 至无数据
项目状态：
已结题

项目摘要

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

丝氨酸途径是微生物同化甲醇等C_1化合物的一条重要途径。该途径包括甲醇脱氢酶（MDH）和丝氨酸羟甲基转移酶（SHMT），二者均能将C_1化合物延长为C_3化合物L-丝氨酸。因此，可以预期在供应甲醇的情况下甘氨酸向L-丝氨酸的转化率为100%。本研究从酶学、蛋白质化学、分子遗传学和晶体学等方面对产丝氨酸的甲醇利用菌Hyphomicrobium methylovorum的丝氨酸途径相关酶，特别是羟丙酮酸还原酶（HPR）进行了研究。发现该酶是由相同亚基（38 kDa）组成的二聚体，该酶的分子量为约70 kDa。该酶在pH值5和9之间在25 ℃下加热10分钟是稳定的。最佳活性w ...更多信息在pH 6.8和45 ℃左右观察到的结果。该酶催化羟基丙酮酸的还原，仅氧化NADH。除了羟基丙酮酸，仅乙醛酸作为底物。发现羟基丙酮酸的Km值为0.175 mM，乙醛酸的Km值为10.8 mM。X-射线晶体学研究表明，HPR是一个对称的二聚体，由分子量为38 kDa的亚基组成，与另一种NAD依赖性酶甲酸脱氢酶具有显著的结构同源性。HPR亚基由两个结构相似的结构域组成，它们通过2重对称性彼此大致相关。这些结构域被深裂分开，形成了潜在的NAD和底物结合位点。其中一个结构域与其它NAD依赖性辅酶A酶的NAD结合结构域结构相似，被鉴定为NAD结合结构域。第二个结构域的拓扑结构与其他酶的各种催化结构域中发现的拓扑结构不同。这是D-特异性2-羟基酸脱氢酶晶体学研究的首次报道。少