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

pentosaceus谷氨酸消旋酶基因由一个795个核苷酸的开放阅读框组成，编码265个氨基酸残基，构成一个单体蛋白。该序列与嗜热葡萄球菌的天冬氨酸消旋酶具有显著的同源性：它不需要辅助因子，并且含有必需的半胱氨酸残基。这两个消旋酶在结构上彼此相似，特别是在两个半胱氨酸残基周围的区域。我们还发现了谷氨酸消旋酶和哺乳动物肌红蛋白之间的显著序列同源性，特别是在肌红蛋白的血红素结合链对应的区域。谷氨酸外消旋酶与等摩尔量的血红蛋白结合使其失活，而天冬氨酸外消旋酶与肌红蛋白序列同源性低，但不与血红蛋白结合。这些提示谷氨酸消旋酶可能起源于球蛋白家族蛋白的进化起源。天冬氨酸外消旋酶也可能是由共同的祖先蛋白进化而来的，但其结构可能比谷氨酸外消旋酶在分化过程中发生了更广泛的改变。我们已经证明，从大肠杆菌中合成d -谷氨酸所需的murI （dga）基因的核苷酸序列推断出的氨基酸序列与P.pentosaceus的谷氨酸消旋酶的氨基酸序列具有显著的同源性。该基因通过设计的核糖体结合位点连接到质粒pKK223-3中，在大肠杆菌JM109细胞中表达。在转化细胞中产生谷氨酸消旋酶，而在宿主细胞中未发现该酶。我部分纯化了酶来表征它。这种酶由两个相同的亚基组成，分子量约为31000。我们还在先前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。这些结果表明，d -谷氨酸是由谷氨酸消旋酶直接由l -对映体产生的，而d -谷氨酸是几乎所有菌株所必需的肽聚糖的组成成分。少

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