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

d -氨基酸氨基转移酶（D-AAT）、丙氨酸消旋酶（AlaR）、宽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d -氨基酸作为细胞壁肽聚糖层的组成部分是细菌必不可少的。这些d -氨基酸在哺乳动物中的生理作用尚不清楚，因此这些酶被认为是开发新型抗菌剂的目标，例如作为自杀底物。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Arg98在D-AAT中的作用，D-AAT是底物α -羧基的结合位点。这可能对酶的独特立体特异性至关重要。用蛋氨酸和赖氨酸替代Arg98，导致氨基酸供体和受体的kmax值降低，Kd值增加。除了上述Arg98突变外，引入另一个突变，即用精氨酸取代位于Arg98附近的Tyr88，导致kmax值增加，但Kd值变化不大。采用遗传互补的方法，将短小芽孢杆菌的谷氨酸消旋酶基因（murl）克隆到d -谷氨酸营养不良菌大肠杆菌WM335中。B.pumilus的MurI编码一个272个氨基酸的蛋白，其起始密码子TTG不寻常，这可能是导致该基因表达效率低的原因。我们还研究了AAR催化的半转和全转，发现该酶催化了非立体特异性的转。

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.