An inducible NADP(+)-dependent D-phenylserine dehydrogenase from Pseudomonas syringae NK-15: purification and biochemical characterization.
An inducible NADP(+)-dependent D-phenylserine dehydrogenase [EC 1.1.1.-], which catalyzes the oxidation of the hydroxyl group of D-threo-beta-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-beta-phenylserine, it utilized D-threo-beta-thienylserine, D-threo-beta-hydroxynorvaline, and D-threonine as substrates but was inert towards other isomers of beta-phenylserine and threonine. It showed maximal activity at pH 10.4 for the oxidation of D-threo-beta-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-beta-phenylserine and NADP+ were 0.44 mM and 29 microM, 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.
Packdibamrung
K
K
Department of Bioresources Science, Kochi University.
Misono
H
H
Harada
M
M
Nagata
S
S
Nagasaki
S
S
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D003067:Coenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009252:NADPH Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D011549:Pseudomonas / Q000201:enzymology*; D012694:Serine / Q000031:analogs & derivatives*; D013379:Substrate Specificity; D013912:Threonine / Q000031:analogs & derivatives*
D001426:Bacterial Proteins; D003067:Coenzymes; D013912:Threonine; D012694:Serine; D000429:Alcohol Oxidoreductases; C086412:phenylserine dehydrogenase (NADP+); D009252:NADPH Dehydrogenase