| Research Abstract |
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.
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