Article Summary
Archaea, 1:133141
© 2002 Heron PublishingVictoria, Canada
contents PDF file subscribe Home page

Molecular analysis of the role of two aromatic aminotransferases and a broad-specificity aspartate aminotransferase in the aromatic amino acid metabolism of Pyrococcus furiosus

DONALD E. WARD,1,2 WILLEM M. DE VOS 1 and JOHN VAN DER OOST 1,3

1 Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands
2 Present address: Department of Chemical Engineering, North Carolina State University, P.O. Box 7905, Raleigh, NC, 27695, USA
3 Author to whom correspondence should be addressed ([email protected])

Received February 14, 2002; accepted June 11, 2002; published online July 12, 2002

Summary
The genes encoding aromatic aminotransferase II (AroAT II) and aspartate aminotransferase (AspAT) from Pyrococcus furiosus have been identified, expressed in Escherichia coli and the recombinant proteins characterized. The AroAT II enzyme was specific for the transamination reaction of the aromatic amino acids, and uses a-ketoglutarate as the amino acceptor. Like the previously characterized AroAT I, AroAT II has highest efficiency for phenylalanine (kcat/Km = 923 s1 mM1). Northern blot analyses revealed that AroAT I was mainly expressed when tryptone was the primary carbon and energy source. Although the expression was significantly lower, a similar trend was observed for AroAT II. These observations suggest that both AroATs are involved in amino acid degradation. Although AspAT exhibited highest activity with aspartate and a-ketoglutarate (kcat ~105 s1), it also showed significant activity with alanine, glutamate and the aromatic amino acids. With aspartate as the amino donor, AspAT catalyzed the amination of a-ketoglutarate, pyruvate and phenylpyruvate. No activity was detected with either branched-chain amino acids or a-keto acids. The AspAT gene (aspC) was expressed as a polycistronic message as part of the aro operon, with expression observed only when the aromatic amino acids were absent from the growth medium, indicating a role in the biosynthesis of the aromatic amino acids.

Keywords
phenylalanine, phenylpyruvate.