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.