权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Structural and functional analysis of bacterial D-amino acid metabolic enzymes to develop their inhibitors

对细菌 D-氨基酸代谢酶进行结构和功能分析以开发其抑制剂

基本信息

批准号：
08456052
负责人：
SODA Kenji
金额：
$ 5.12万
依托单位：
Kansai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08456052/
关键词：
D-amino acid aminotransafearse alanine racemase peptidoglycan glutamate racemase reaction mechanism D-アミノ酸

项目摘要

D-Amino acid aminotransferase (D-AAT), alanine racemase (AlaR), amino acid racemase of broad substrate specificities (AAR), and glutamate racemase (GluR) participate in the metabolism of D-amino acids, some of which are indispensable for bacteria as components of the peptidoglycan layr of cell walls. Physiological roles of these D-amino acids in mammals are not known, thus these enzymes have been regarded as a target for the development of novel antibacterial agents serving, for example, as suicide substrates. To develop the mechanism-based inhibitors, we studied the structure-function relationship of these enzymes. We studied the role of Arg98 of D-AAT,the binding site for the alpha-carboxyl group of substrates. This is presumably crucial for the unique stereospecificity of the enzyme. Replacement of Arg98 by replacement by methionine and lysine, resulted in decreases in the kmax values and increases in the Kd values for both amino donors and amino acceptors. The introduction of another mutation, replacement of Tyr88 being located near Arg98 by arginine, in addition to the above Arg98 mutation, resulted in increases in the kmax values but little change in the Kd values. We cloned the glutamate racemase gene (murl) of Bacillus pumilus cells into E.coli WM335, a D-glutamate auxotroph, by means of a genetic complement method. MurI of B.pumilus encodes a 272-amino acid protein with an unusual initiation codon, TTG,which is probably the reason for the low expression efficiency of the gene. We also studied the half and overall transaminations catalyzed by AAR,and found that the enzyme catalizes the non-stereospecific transaminations.

D-氨基酸转氨酶 (D-AAT)、丙氨酸消旋酶 (AlaR)、广泛底物特异性氨基酸消旋酶 (AAR) 和谷氨酸消旋酶 (GluR) 参与 D-氨基酸的代谢，其中一些氨基酸作为细胞壁肽聚糖层的组成部分对于细菌来说是不可或缺的。这些 D-氨基酸在哺乳动物中的生理作用尚不清楚，因此这些酶已被视为开发新型抗菌剂的靶标，例如用作自杀底物。为了开发基于机制的抑制剂，我们研究了这些酶的结构-功能关系。我们研究了 D-AAT 中 Arg98 的作用，D-AAT 是底物 α-羧基的结合位点。这可能对于酶独特的立体特异性至关重要。用甲硫氨酸和赖氨酸替换 Arg98 导致氨基供体和氨基受体的 kmax 值降低和 Kd 值增加。除了上述 Arg98 突变之外，引入另一种突变，即用精氨酸替换位于 Arg98 附近的 Tyr88，导致 kmax 值增加，但 Kd 值变化不大。我们通过遗传补体方法将短小芽孢杆菌细胞的谷氨酸消旋酶基因（murl）克隆到D-谷氨酸营养缺陷型大肠杆菌WM335中。 B.pumilus的MurI编码一个272个氨基酸的蛋白质，其起始密码子TTG不寻常，这可能是该基因表达效率低的原因。我们还研究了AAR催化的半转氨作用和整体转氨作用，发现该酶催化非立体特异性转氨作用。