Purification and characterization of the alanine aminotransferase from thehyperthermophilic archaeon Pyrococcus furiosus and its role in alanine production

Alanine aminotransferase (ALaAT) was purified from cell extracts of the hyp erthermophilic archaeon Pyrococcus furiosus by multistep chromatography. Th e enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel fi ltration, and consists of two identical subunits of 46 kDa, as deduced by s odium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene seque nce. The AlaAT displayed a broader substrate specificity than AlaATs from e ukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor . Optimal activity was found in the pH range of 6.5 to 7.8 and at a tempera ture of over 95 degrees C. The N-terminal amino acid sequence of the purifi ed AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in the P. furiosus genome database. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. The pH and tempe rature dependence, molecular mass, and kinetic parameters of the recombinan t were indistinguishable from those of the native enzyme from P. furiosus. The k(cat)/K-m values for alanine and pyruvate formation were 41 and 33 s(- 1) mM(-1), respectively, suggesting that the enzyme is not biased toward ei ther the formation of pyruvate, or alanine, Northern analysis identified a single 1.2-kb transcript for the aat gene. In addition, both the aat and gd h (encoding the glutamate dehydrogenase) transcripts appear to be coregulat ed at the transcriptional level, because the expression of both genes was i nduced when the cells were grown on pyruvate. The coordinated control found for the aat and gdh genes is in good agreement with these enzymes acting i n a concerted manner to form an electron sink in P. furiosus.