Purification and characterization of serine racemase from a hyperthermophilic archaeon, Pyrobaculum islandicum.
Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100 degrees C. A pyridoxal 5'-phosphate-dependent serine racemase called Srr was purified from the organism. The corresponding srr gene was cloned, and recombinant Srr was purified from Escherichia coli. It showed the highest racemase activity toward L-serine, followed by L-threonine, D-serine, and D-threonine. Like rodent and plant serine racemases, Srr is bifunctional, showing high L-serine/L-threonine dehydratase activity. The sequence of Srr is 87% similar to that of Pyrobaculum aerophilum IlvA (a putative threonine dehydratase) but less than 32% similar to any other serine racemases and threonine dehydratases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses revealed that Srr is a homotrimer of a 44,000-molecular-weight subunit. Both racemase and dehydratase activities were highest at 95 degrees C, while racemization and dehydration were maximum at pH 8.2 and 7.8, respectively. Unlike other, related Ilv enzymes, Srr showed no allosteric properties: neither of these enzymatic activities was affected by either L-amino acids (isoleucine and valine) or most of the metal ions. Only Fe2+ and Cu2+ caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. The Km and Vmax values of the racemase activity of Srr for L-serine were 185 mM and 20.1 micromol/min/mg, respectively, while the corresponding values of the dehydratase activity of L-serine were 2.2 mM and 80.4 micromol/min/mg, respectively.
Ohnishi
Masato
M
Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-14 Kanda-Surugadai, Chiyoda Ward, Tokyo 101-8308, Japan.
Saito
Makoto
M
Wakabayashi
Sadao
S
Ishizuka
Morio
M
Nishimura
Katsushi
K
Nagata
Yoko
Y
Kasai
Sabu
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000494:pharmacology; D008957:Models, Genetic; D008969:Molecular Sequence Data; D041543:Pyrobaculum / Q000201:enzymology* / Q000235:genetics; D019998:Racemases and Epimerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D000099300:Serine Racemase
D000255:Adenosine Triphosphate; D019843:Archaeal Proteins; D007477:Ions; D019998:Racemases and Epimerases; D011994:Recombinant Proteins; D012694:Serine; D013912:Threonine; D000099300:Serine Racemase