title: Highly selective L-threonine 3-dehydrogenase from Cupriavidus necator and its use in determination of L-threonine.
abstract: l-Threonine level in blood plasma is a biomarker of some diseases and nitrogen imbalance in the body. The determination of l-threonine is interesting and is required for diagnosis and management of inherited metabolic disorder. This is the first report of the specific enzymatic determination of l-threonine by a newly discovered l-threonine 3-dehydrogenase (ThrDH, EC 1.1.1.103) from Cupriavidus necator NBRC 102504. ThrDH, a key enzyme in l-threonine catabolism in microorganisms and animals, catalyzes the NAD(+)-dependent oxidation of l-threonine to 2-amino-3-oxobutyrate. ThrDH from C. necator was purified to homogeneity and fully characterized. l-Threonine and dl-2-amino-3-hydroxyvalerate are the only substrates for ThrDH among other l-amino acids, alcohols, and amino alcohols. The primary amino acid structure of ThrDH belongs to the extended short-chain alcohol dehydrogenase superfamily and is related to GDP-mannose-3',5'-epimerase (GME) from Arabidopsis thaliana. Both enzymes have a glycine-rich NAD(+)-binding domain at the N terminal and conserved catalytic triad of YxxxK residues, but substrate-binding residues of GME were not found in the ThrDH sequence. ThrDH significantly differs from known bacterial and archaea ThrDHs that belong to zinc-binding medium chain alcohol dehydrogenase because of low sequence similarity and the lack of a zinc-binding domain in the sequence. A specific, quantitative, and sensitive enzymatic endpoint method for l-threonine determination was developed by using a ThrDH microplate assay. The assay was successfully applied for determination of l-threonine in human serum and plasma. Our specific determination is simple, convenient, inexpensive, accurate, and suitable for mass screening determination of l-threonine in a number of samples.
authors.affiliation: Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Techawaree
authors.initials: T
authors.lastname: Ueatrongchit
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Asano
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques / Q000191:economics / Q000379:methods*; D002417:Cattle; D002474:Cell-Free System / Q000201:enzymology; D003001:Cloning, Molecular; D020118:Cupriavidus necator / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013696:Temperature; D013912:Threonine / Q000032:analysis* / Q000097:blood / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D008670:Metals; D013912:Threonine; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase