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

松材线虫谷氨酸消旋酶基因全长795个核苷酸，编码265个氨基酸残基，形成一个单体蛋白。该序列与嗜热链霉菌的天冬氨酸外消旋酶有显著的同源性：它不需要辅因子，并且含有一个必需的半胱氨酸基残基。这两个外消旋酶在结构上彼此相似，特别是在两个半胱氨基残基周围的区域。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，特别是在与肌红蛋白的血红素结合链相对应的区域。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。这些结果表明，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。由于分化，其结构可能比谷氨酸消旋酶发生了更广泛的改变。我们已经证明，由MURI(DGA)基因的核苷酸序列推导出的氨基酸序列与戊糖酵母的谷氨酸消旋酶有显著的同源性，该基因是合成D-谷氨酸的必需成分。将该基因连接到含有核糖体结合位点的pKK223-3载体上，并在E.coliJM109细胞中表达。转化细胞中产生了谷氨酸消旋酶，而宿主细胞中未发现该酶。我对这种酶进行了部分纯化，以确定其特性。该酶由两个相同的亚基组成，分子量约为31,000。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细菌的染色体上。这表明D-谷氨酸是由其L对映体通过谷氨酸外消旋酶直接合成的，是几乎所有细菌不可缺少的肽聚糖组分。较少

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