An Inducible NADP+-Dependent D-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15: Purification and Biochemical Characterization

• Published in: Journal of biochemistry (Tokyo) 1993-12, Vol.114 (6), p.930-935
• Main Authors: Packdibamrung, Kanoktip, Misono, Haruo, Harada, Makoto, Nagata, Shinji, Nagasaki, Susumu
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases - antagonists & inhibitors; Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - isolation & purification; Amino Acid Sequence; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Coenzymes - antagonists & inhibitors; Coenzymes - chemistry; Coenzymes - isolation & purification; Hydrogen-Ion Concentration; Isomerism; Kinetics; Molecular Sequence Data; Molecular Weight; NADPH Dehydrogenase - antagonists & inhibitors; NADPH Dehydrogenase - chemistry; NADPH Dehydrogenase - isolation & purification; Pseudomonas - enzymology; Serine - analogs & derivatives; Substrate Specificity; Threonine - analogs & derivatives
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a124279

An inducible NADP+-dependent D-phenylserine dehydrogenase [EC 1.1.1.-], which catalyzes the oxidation of the hydroxyl group of D-threo-β-phenylserine, was purified to homogeneity from a crude extract of Pseudomonas syringae NK-15 isolated from soil. The enzyme consisted of two subunits identical in molecular weight (about 31, 000). In addition to D-threo-β-phenylserine, it utilized D-threo-β-thienylserine, D-threo-β-hydroxynor-valine, and D-threonine as substrates but was inert towards other isomers of β-phenyl-serine and threonine. It showed maximal activity at pH 10.4 for the oxidation of D-threo-β-phenylserine, and it required NADP+ as a natural coenzyme. NAD+ showed a slight coenzyme activity. The enzyme was inhibited by p-chloromercuribenzoate, HgCl2, and monoiodoacetate but not by the organic acids such as tartronate. The Michaelis constants for D-threo-β-phenylserine and NADP+ were 0.44 mM and 29 μM, respectively. The N-terminal 27 amino acids sequence was determined. It suggested that the NADP+-binding site was located in the N-terminal region of the enzyme.