RRC ID 11447
著者 Ueatrongchit T, Asano Y.
タイトル Highly selective L-threonine 3-dehydrogenase from Cupriavidus necator and its use in determination of L-threonine.
ジャーナル Anal Biochem
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.
巻・号 410(1)
ページ 44-56
公開日 2011-3-1
DOI 10.1016/j.ab.2010.11.003
PII S0003-2697(10)00716-5
PMID 21073854
MeSH Alcohol Oxidoreductases / chemistry Alcohol Oxidoreductases / genetics Alcohol Oxidoreductases / isolation & purification Alcohol Oxidoreductases / metabolism* Amino Acid Sequence Animals Biosensing Techniques / economics Biosensing Techniques / methods* Cattle Cell-Free System / enzymology Cloning, Molecular Cupriavidus necator / enzymology* Enzyme Inhibitors / pharmacology Escherichia coli / genetics Humans Hydrogen-Ion Concentration Metals / pharmacology Mice Molecular Sequence Data Substrate Specificity Temperature Threonine / analysis* Threonine / blood Threonine / metabolism
IF 2.877
引用数 13
WOS 分野 BIOCHEMICAL RESEARCH METHODS CHEMISTRY, ANALYTICAL BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
一般微生物 JCM 5909 JCM 20909