ABSTRACT
D-Threonine dehydrogenase (EC 1.1.1) catalyses the oxidation of the 3-hydroxyl group of D-threonine. The nucleotide sequence of the structural gene, dtdS, for this enzyme from Pseudomonas cruciviae IFO 12047 was determined. The dtdS gene encodes a 292 amino acid polypeptide. The enzyme was overproduced in Escherichia coli cells; the activity was found in cell extracts of the clone. The enzyme showed high sequence similarity to 3-hydroxyisobutyrate dehydrogenases. This is the first example showing the primary structure of an enzyme catalysing the NADP(+)-dependent dehydrogenation of D-threo-3-hydroxyamino acids.
Subject(s)
Alcohol Oxidoreductases/genetics , Genes, Bacterial , Pseudomonas/enzymology , Pseudomonas/genetics , Alcohol Oxidoreductases/chemistry , Alcohol Oxidoreductases/metabolism , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA Primers/genetics , Escherichia coli/genetics , Gene Expression , Molecular Sequence Data , Molecular Weight , Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Alcohol Oxidoreductases/isolation & purification , Bacterial Proteins/isolation & purification , NADPH Dehydrogenase/isolation & purification , Pseudomonas/enzymology , Serine/analogs & derivatives , Threonine/analogs & derivatives , Alcohol Oxidoreductases/antagonists & inhibitors , Alcohol Oxidoreductases/chemistry , Amino Acid Sequence , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , 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 , Substrate SpecificityABSTRACT
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.
