title: NADP(+)-dependent D-threonine dehydrogenase from Pseudomonas cruciviae IFO 12047.
abstract: NADP(+)-dependent D-threonine dehydrogenase (EC 1.1.1.-), which catalyzes the oxidation of the 3-hydroxyl group of D-threonine, was purified to homogeneity from a crude extract of Pseudomonas cruciviae IFO 12047. The enzyme had a molecular mass of about 60,000 Da and consisted of two identical subunits. In addition to D-threonine, D-threo-3-phenylserine, D-threo-3-thienylserine, and D-threo-3-hydroxynorvaline were also substrates. However, the other isomers of threonine and 3-phenylserine were inert. The enzyme showed maximal activity at pH 10.5 for the oxidation of D-threonine. The enzyme required NADP+. NAD+ showed only slight activity. The enzyme was not inhibited by EDTA, o-phenanthroline, alpha,alpha'-dipyridyl, HgCl2, or p-chloromercuribenzoate but was inhibited by tartronate, malonate, pyruvate, and DL-2-hydroxybutyrate. The inhibition by these organic acids was competitive against D-threonine. Initial-velocity and product inhibition studies suggested that the oxidation proceeded through a sequential ordered Bi Bi mechanism. The Michaelis constants for D-threonine and NADP+ were 13 and 0.12 mM, respectively.
authors.affiliation: Department of Bioresources Science, Kochi University, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Misono
authors.forename: I
authors.initials: I
authors.lastname: Kato
authors.forename: K
authors.initials: K
authors.lastname: Packdibamrung
authors.forename: S
authors.initials: S
authors.lastname: Nagata
authors.forename: S
authors.initials: S
authors.lastname: Nagasaki
mesh_terms: D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013912:Threonine / Q000031:analogs & derivatives
chemical_list: D013912:Threonine; D009249:NADP; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase