Highly thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Thermococcus kodakaraensis.
l-Threonine dehydrogenase, a key enzyme in the l-threonine metabolism, catalyses the NAD(+)-dependent conversion of l-threonine to 2-amino-3-ketobutyrate, that non-enzymically decarboxylates to aminoacetone. A search of the genome sequence of hyperthermophilic archaeon, Thermococcus kodakaraensis revealed the presence of a closely related orthologue (TK0916) of archaeal and bacterial l-threonine dehydrogenase genes. Expression in Escherichia coli, purification and characterization of the TK0916 gene product revealed that this gene actually coded for a protein with high levels of l-threonine dehydrogenase activity (7.26 U mg(-1)). The enzyme exhibited highest activity at pH 12 and 90 degrees C. The K(m) values for l-threonine and NAD(+) at 50 degrees C were 1.6 mM and 0.028 mM, respectively. The enzyme activity was dependent on divalent cations. The half-life of the enzyme was more than 2 h at 85 degrees C and 24 min in boiling water. This is the most thermostable threonine dehydrogenase exhibiting optimal activity at the highest pH (12) reported to date. This is the first report on the characterization of a TDH from genus Thermococcus.
Bashir
Qamar
Q
University of the Punjab, Lahore, Pakistan.
Rashid
Naeem
N
Jamil
Farrukh
F
Imanaka
Tadayuki
T
Akhtar
Muhammad
M
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006207:Half-Life; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics; D015032:Zinc / Q000494:pharmacology
D019843:Archaeal Proteins; D008670:Metals; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase; D015032:Zinc