A STEREO-INVERTING D-PHENYLGLYCINE AMINOTRANSFERASE FROM PSEUDOMONAS-STUTZERI ST-201 - PURIFICATION, CHARACTERIZATION AND APPLICATION FOR D-PHENYLGLYCINE SYNTHESIS

D-phenylglycine aminotransferase (D-PhgAT) from a newly isolated soil bacterium, Pseudomonas stutzeri ST-201, was purified to electrophoreti c homogeneity and characterized. The molecular weight (M-r) of the nat ive enzyme was estimated to be 92000. It is composed of two subunits i dentical in molecular weight (M-r=47500). The isoelectric point (pI) o f the native enzyme was 5.0. The enzyme catalyzed reversible transamin ation specific for D-phenylglycine or D-4-hpdroxyphenylglycine in whic h 2-oxoglutarate was an exclusive amino group acceptor and was convert ed into L-glutamic acid. Neither the D- nor L-isomer of phenylalanine, tyrosine, alanine, valine, leucine, isoleucine or serine could serve as a substrate. The enzyme was most active at alkaline pH with maximum activity at pH 9-10. The temperature for maximum activity was 35-45 d egrees C. The apparent K-m values for D-phenylglycine and for 2-oxoglu tarate at 35 degrees C, pH 9.5 were 1.1 and 2.4 mM, respectively. The enzyme activity was strongly inhibited by typical inhibitors of pyrido xal phosphate-dependent enzymes. Possible application of this enzyme f or synthesis of enantiomerically pure D-phenylglycine was demonstrated . (C) 1997 Elsevier Science B.V.