An extremely thermostable aromatic aminotransferase from the hyperthermophilic archaeon Pyrococcus furiosus

Abstract

Pyrococcus furiosus is a strictly anaerobic archaeon (formerly archaebacterium) that grows optimally at 100°C by the fermentation of peptides. Cell-free extracts were found to contain two distinct aromatic aminotransferases (ArAT, EC 2.6.1.57), one of which was purified to electrophoretic homogeneity. P. furiosus ArAT is a homodimer with a subunit M r value of 44 000 ± 1000. Using 2-ketoglutarate as the amino acceptor, the purified enzyme catalyzed the pyridoxal 5′-phosphate (PM P)-dependent transamination of phenylalanine, tyrosine and tryptophan with respective k cat values of 253, 72 and 62 (s −1 at 80°C) under saturating conditions. The K m values for all three amino acids were between 1.1 and 2.1 mM and the optimum temperature for catalysis was above 95°C. The melting point for the pure enzyme was also above 95°C as determined by change in ellipticity at 220 nm. Irreversible denaturation of the pure enzyme was not apparent after 6 h at 80°C in the presence of PM P and 2-ketoglutarate and the time required for a 50% loss in activity at 95°C was approx. 16 h. This decreased to approx. 12 h if cofactor and substrate were not added. In contrast, the apoenzyme (lacking PM P) lost most (70%) of its activity (measured after reconstitution) after 6 h at 80°C, indicating that both PM P and 2-ketoglutarate stabilize the enzyme at extreme temperatures. Although few ArATs have been characterized to date, the molecular properties and substrate specificity of P. furiosus ArAT more resemble those of the ArAT from Escherichia coli than those of the analogous enzyme from rat liver. Moreover, the P. furiosus enzyme is by far the most thermostable aminotransferase of any type to be purified so far.