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Biochemical and Molecular Biological Studies or Structure and Function of Glutamate Racemase

谷氨酸消旋酶的生化和分子生物学研究或结构和功能

基本信息

批准号：
06680610
负责人：
ESAKI Nobuyoshi
金额：
$ 1.41万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06680610/
关键词：
glutamate racemase gene cloning Bacillus cereus cysteine residue composite active site alpha, beta-elimination suicide substrate serine O-sulfate composite active site

项目摘要

The glutamate racemase gene of P.pentosaceus consists of a 795-nucleotides open reading frame, and encodes 265 amino acid residues which form a monomeric protein. The sequence shows significant homology with that of aspartate racemase from S.thermophilus : it requires no cofactors and contains an essential cysteinyl residue. The two racemases are structurally similar to each other, in particular in the regions around the two cysteinyl residues. We have also found significant sequence homology between glutamate racemase and mammalian myoglobins, in particular in the regions corresponding to a heme binding procket of myoglobins. Glutamate racemase is bound with an equimolar amount of hemin to be inactivated, and aspartate racemase shows a low sequence homology with myoglobins, but is not bound with hemin. These suggest that glutamate racemase may be derived from an evolutionary origin of globin family proteins. Aspartate racemase also may have evolved from the common ancestral protein, b … More ut its structure may have been altered more extensively than glutamate racemase by divergence. We have shown that the amino acid sequence deduced from the nucleotide sequence of murI (dga) gene that is required for the biosynthesis of D-glutamate as an essential component of peptidoglycan in E.coli has significant homology with that of glutamate racemase of P.pentosaceus. The gene was ligated into a plasmid, pKK223-3 with a designed ribosome binding site, and expressed in E.coli JM109 cells. Glutamate racemase was produced in the transformant cells, whereas the enzyme was not found in the host cells. I partially purified the enzyme to characterize it. The enzyme consists of two identical subunits with a molecular weight of about 31,000. We have also found three highly conserved regions in the glutamate racemase genes previously sequenced and shown that their conserved regions exsist on the chromosomes of various kinds of bacterial strains. These indicate that D-glutamate, which is indispensable for almost all bacterial strains as a constituent of peptidoglycans, is directly produced from its L-enantiomer by glutamate racemase. Less

戊糖青霉谷氨酸消旋酶基因由795个核苷酸的开放阅读框组成，编码265个氨基酸残基，形成单体蛋白。该序列与来自嗜热链球菌的天冬氨酸消旋酶显示出显著的同源性：它不需要辅因子并且含有必需的半胱氨酰残基。这两种消旋酶在结构上彼此相似，特别是在两个半胱氨酰残基周围的区域中。我们还发现谷氨酸消旋酶和哺乳动物肌红蛋白之间的显着序列同源性，特别是在对应于肌红蛋白的血红素结合链的区域。谷氨酸消旋酶与等摩尔量的氯化血红素结合而失活，天冬氨酸消旋酶与肌红蛋白序列同源性低，但不与氯化血红素结合。这表明谷氨酸消旋酶可能来源于珠蛋白家族蛋白的进化起源。天冬氨酸消旋酶也可能是从共同的祖先蛋白B进化而来的 ...更多信息但它的结构可能比谷氨酸消旋酶因趋异而发生更广泛的改变。我们已经表明，推导出的氨基酸序列的核苷酸序列的murI（dga）基因，所需的D-谷氨酸的生物合成的肽聚糖在大肠杆菌中具有显着的同源性与戊糖丙酸杆菌的谷氨酸消旋酶。将该基因连接到具有设计的核糖体结合位点的质粒pKK 223 -3中，并在大肠杆菌JM 109细胞中表达。谷氨酸消旋酶产生于大肠杆菌细胞中，而在宿主细胞中没有发现该酶。我对这种酶进行了部分纯化以确定其特性，这种酶由两个相同的亚基组成，分子量约为31000。在已测序的谷氨酸消旋酶基因中发现了三个高度保守的区域，并表明它们的保守区域存在于各种菌株的染色体上。这表明，D-谷氨酸，这是必不可少的几乎所有的细菌菌株作为肽聚糖的组成部分，是直接产生的L-对映体的谷氨酸消旋酶。少