Structure and Function of Enzymes Participating in Metabolism of D-Amino Acid of Bacterial Cell Walls and Development of their Specific Inhibitors

D-氨基酸，如D-丙氨酸和D-谷氨酸，是细菌细胞壁肽聚糖的不可忽视的成分。因此，参与D-氨基酸生物合成的细菌酶的特异性抑制剂可能是有效的抗生素。我们对丙氨酸消旋酶、D-氨基酸转氨酶和谷氨酸消旋酶的作用机理进行了详细的研究，以期开发出这些酶的消旋酶抑制剂。我们研究了嗜热脂肪芽孢杆菌耐热丙氨酸外消旋酶Lys39与辅因子5‘-磷酸吡哆醛(PLP)结合的作用，并认为该残基可能是定点突变的催化碱基。Lys39的末端氨基是从底物中提取2-氢的碱基。烷基胺对K39A突变酶的影响表明，Lys39作为唯一的催化碱从底物中提取2-氢，并将其返回到去质子化的中间体…的底物部分的2-碳吃得更多了。D-氨基酸转氨酶催化各种D-氨基酸和酮酸之间的氨基转移。我们研究了耐热的D-氨基酸转氨酶的亮氨酸201残基的催化作用：通过定点突变，发现亮氨酸201残基位于活性中心附近，与结合的PLP相互作用。Leu 201残基可能在PMP转化为PLP的过程中调节辅因子的作用，谷氨酸消旋酶催化谷氨酸外消旋生成D-谷氨酸。我们将该酶与乳酸菌的酶进行了比较。加入大肠杆菌酶的激活剂UDP-N-乙酰胞壁酰胺-L-丙氨酸影响了该酶的CD光谱和荧光光谱。反之，N-乙酰菌胺-L-丙氨酸不能激活戊糖对虾和短对虾的酶活性。链球菌酶与哺乳动物肌红蛋白有显著的序列相似性，并被氯化血红素抑制。氯化高铁血红素在无UDP-N-乙酰菌胺-L-丙氨酸时对大肠杆菌的谷氨酸外消旋酶无抑制作用，但在UDP-N-乙酰菌胺-L-丙氨酸存在时对其有较强的抑制作用。通过添加UDP-N-乙酰胞壁酰基-L-丙氨酸，将大肠杆菌酶的构象转化为类似于小球菌酶的构象。较少

D-Amino acids such as D-alanine and D-glutamate are the indespensable components of the peptidoglycan layr of the bacterial cell walls. Thus, the specific inhibitors for the bacteral enzymes participate in the biosyntheses of D-amino acids can be potent antibiotics. We studied detailed mechanisms of alanine racemase, D-amino acid aminotransferase, and glutamate racemase, to develop the meachanism-based inhibitors for these enzymes. We studied the role of Lys39 of the thermostable alanine racemase of Bacillus stearothermophilus, which is bound to the cofactor, pyridoxal 5'-phosphate (PLP) , and suggested this residue to be a catalytic base by site-directed mutagenesis. The terminal amino group of Lys39 acts as a base to abstract the 2-hydrogen from the substrate. The effects of alkylamines on the K39A mutant enzymes suggest that Lys39 acts as a sole catalytic base to abstract the 2-hydrogen from the substrate and returns it to the 2-carbon of substrate moiety of a deprotonated intermedi … More ate. D-Amino acid aminotransferase catalyzes the transfer of amino group between various D-amino acids and keto acids. We studied the catalytic role of leucine 201 residue of the themostable D-amino acid aminotransferase : the residue was crystallographically shown to be in the vicinity of the active-site to interact with the bound PLP by site-directed mutagenesis. The Leu 201 residue probably regulates the function of cofactor during the conversion of PMP to PLP.Glutamate racemase catalyzes the racemization of glutamate to produce D-glutamate. We compared the enzyme with those of Lactic acid bacteria. The addition of UDP-N-acetylmuramyl-L-alanine, an activator of the E.coli enzyme affected the CD and fluorescence spectra of the E.coli enzyme. In contrast, the enzymes of P.pentosaceus and L.brevis were not activated by UDP-N-acetylmuramyl-L-alanine. The Pediococcus enzyme shows a significant sequence similarity to mammalian myoglobin, and was inhibited by hemin. Glutamate racemase of E.coli was not inhibited by hemin in the absence of UDP-N-acetylmuramyl-L-alanine, but strongly inhibited in the presence of UDP-N-acetylmuramyl-L-alanine. The conformation of the E.coli enzyme is converted to a similar form to that of the Pediococcus enzyme by the addition of UDP-N-acetylmuramyl-L-alanine. Less