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C_1化合物（如甲基苯甲醇）的特征微生物同化途径。该途径包括甲基戊二醇脱氢酶（MDH）和丝氨酸羟基转移酶（SHMT），通过将C_1化合物延长至C_3化合物L链，l塞链。这是可以预期，甲基苯二甲醇供应的转化率为100％的甘氨酸到L丝氨酸。本研究揭示了与丝氨酸途径相关的酶，蛋白质化学，分子遗传和晶体学阐明丝氨酸途径，尤其是丝氧酸酯还原（HPR），丝氨酸产生的甲基苯酚 - 二甲基二氧化甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基植物的甲基甲基甲基植物的甲基植物疾病综合孔植物，第一次从甲基营养中获得酶。发现该酶是由相同亚基（38 kDa）组成的二聚体，酶的分子质量约为70 kDa。该酶在5和9之间的pH值25 c处的加热稳定。最佳活性W…在pH 6.8和45^-c大约45^-c左右观察到。酶催化了羟基扭转的降低，仅氧化NADH的氧化，仅是NADH的氧化。其他仅是羟基甲甲基甲酰甲酰甲酰胺，仅用于甲氧甲甲基盐酸盐。发现羟基丙酮酸的Km值为0.175 mm，乙二醇的值为10.8mm。 HPR的X射线晶体学研究表明，该酶分子是由38KDA的分子质量亚基组成的对称二聚体，并且与另一种NAD依赖性酶形成脱氢酶。 HPR亚基由两个结构上相似的域组成，它们近似相互关联，通过2倍对称性。这些结构域被形成纯化的NAD和底物结合位点的深裂裂。其中一个结构域被确定为NAD结合结构域，其基于其其他NAD依赖性脱氢酶的NAD结合结构域的紧密结构相似性。第二结构域的拓扑与其他脱氢酶的各种催化结构域的拓扑不同。这是D特异性2-羟基酸脱氢酶的晶体学研究的第一个报告。较少的