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

细菌细胞壁D-氨基酸代谢酶的结构和功能及其特异性抑制剂的开发

• 批准号: 06454077
• 负责人: SODA Kenji
• 金额: $ 5.06万
• 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 (B)
• 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-06454077/
• 关键词: D-Amino acid; Alanine Racemase; D-Amino acid aminotransferase; Glutamate racemase; Suicide substrate; 酵素自殺基質; UDP-N-アセチルムラミル-L-アラニン; ピリドキサルリン酸; D-アミノ酸トランスアミナーゼ; セリンO-スルフェート; 酵素阻害剤

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

D-氨基酸如D-丙氨酸和D-谷氨酸是细菌细胞壁的肽聚糖层的不可缺少的组分。因此，参与D-氨基酸生物合成的细菌酶的特异性抑制剂可以是有效的抗生素。本论文对丙氨酸消旋酶、D-氨基酸氨基转移酶和谷氨酸消旋酶的作用机制进行了详细的研究，旨在开发基于机制的酶抑制剂。我们研究了嗜热脂肪芽孢杆菌的热稳定丙氨酸消旋酶的Lys 39的作用，该酶与辅因子吡哆醛5 '-磷酸（PLP）结合，并通过定点突变提出该残基是催化碱基。Lys 39的末端氨基作为碱从底物中提取2-氢。烷基胺对K39 A突变酶的影响表明，Lys 39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中间体的底物部分的2-碳。 ...更多信息 吃过了D-氨基酸氨基转移酶催化各种D-氨基酸和酮酸之间的氨基转移。我们研究了热稳定D-氨基酸氨基转移酶的亮氨酸201残基的催化作用：该残基在晶体学上显示在活性位点附近，通过定点突变与结合的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功能。谷氨酸消旋酶催化谷氨酸消旋生成D-谷氨酸。我们比较了这些酶与乳酸菌。加入UDP-N-乙酰胞壁酰-L-丙氨酸（大肠杆菌酶的激活剂）影响大肠杆菌酶的CD和荧光光谱。相反，戊糖原杆菌和短乳杆菌的酶不被UDP-N-乙酰胞壁酰-L-丙氨酸激活。片球菌酶显示出与哺乳动物肌红蛋白的显著序列相似性，并且被氯化血红素抑制。在不存在UDP-N-乙酰胞壁酰-L-丙氨酸的情况下，氯化血红素不抑制大肠杆菌的谷氨酸消旋酶，但在存在UDP-N-乙酰胞壁酰-L-丙氨酸的情况下强烈抑制谷氨酸消旋酶。通过添加UDP-N-乙酰胞壁酰-L-丙氨酸，大肠杆菌酶的构象被转化为与片球菌酶的构象相似的形式。少

项目成果

K.Kishimoto et al.: "Role of Leucine 201 of Thermostable D-Amino Acid Aminotrasferase from a Thermophile." J.Biochem.(印刷中). (1994)

K.Kishimoto 等人：“来自嗜热菌的热稳定 D-氨基酸氨基转移酶的亮氨酸 201 的作用”。J.Biochem（出版中）。

Ashiuchi Makoto: "In Vivo Effect of GroESL on the Folding of Glutamate Racemase of Eschirichia coli." J.Biochem.117. 495-498 (1995)

Ashiuchi Makoto：“GroESL 对大肠杆菌谷氨酸消旋酶折叠的体内影响。”

H. Toyama et al.: "Reconstitute of Fragmentary Form of Thermostable Alanine Racemase" Biosci. Biotech. Biochem.59. 1118-1120 (1995)

H. Toyama 等人：“热稳定丙氨酸消旋酶片段形式的重构”Biosci。

Nishimura Katsushi: "A Simple Method for Detemination of Stereospecificity of Aminotransferase for C-4′Hydrogen Transfer of the Coenzyme." Bioorganic & Medical Chemistry. 2. 605-607 (1994)

Katsushi Nishimura：“测定辅酶 C-4 氢转移的氨基转移酶立体特异性的简单方法。”2. 605-607 (1994)

Choi, Soo-Young: "Bacterial Glutamate Racemase has High Sequence Homology with Myoglobins and Forms an Equimolar Inactive Complex with Hemin." Proc.Natl.Acad.Sci., USA. 91. 10144-10147 (1994)

Choi, Soo-Young：“细菌谷氨酸消旋酶与肌红蛋白具有高序列同源性，并与氯化血红素形成等摩尔的非活性复合物。”