Purification and Characterization of Serine Racemase from a Hyperthermophilic Archaeon, Pyrobaculum islandicum

Abstract

ABSTRACT Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100°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°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 Fe 2+ and Cu 2+ caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. The K m and V max values of the racemase activity of Srr for l -serine were 185 mM and 20.1 μmol/min/mg, respectively, while the corresponding values of the dehydratase activity of l -serine were 2.2 mM and 80.4 μmol/min/mg, respectively.