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 electrophoretic homogeneity and characterized. The molecular weight (M(r)) of the native enzyme was estimated to be 92,000. It is composed of two subunits identical in molecular weight (M(r)) = 47,500). The isoelectric point (pI) of the native enzyme was 5.0. The enzyme catalyzed reversible transamination specific for D-phenylglycine or D-4-hydroxyphenylglycine in which 2-oxoglutarate was an exclusive amino group acceptor and was converted 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 degrees C. The apparent K(m) values for D-phenylglycine and for 2-oxoglutarate 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 pyridoxal phosphate-dependent enzymes. Possible application of this enzyme for synthesis of enantiomerically pure D-phenylglycine was demonstrated.
Wiyakrutta
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S
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Meevootisom
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V
D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors; D018698:Glutamic Acid / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D006038:Glyoxylates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007656:Ketoglutaric Acids / Q000378:metabolism; D008333:Mandelic Acids; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature; D000637:Transaminases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D004791:Enzyme Inhibitors; D006038:Glyoxylates; D007656:Ketoglutaric Acids; D008333:Mandelic Acids; C012482:phenylglyoxylic acid; D018698:Glutamic Acid; C008852:2-phenylglycine; C023010:4-hydroxyphenylglycine; C108039:D-phenylglycine aminotransferase; D000637:Transaminases; D005998:Glycine