Thermotoga neapolitana adenylate kinase is highly active at 30 degreesC

Abstract

The adenylate kinase (AK) gene from Thermotoga neapolitana, a hyperthermophilic bacterium, was cloned and overexpressed in Escherichia coli, and the recombinant enzyme was biochemically characterized. The T. neapolitana AK (TNAK) sequence indicates that this enzyme belongs to the long bacterial AKs. TNAK contains the four cysteine residues that bind Zn2+ in all Gram-positive AKs and in a few other Zn2+-containing bacterial AKs. Atomic emission spectroscopy and titration data indicate a content of 1 mol of Zn2+/mol of recombinant TNAK. The EDTA-treated enzyme has a melting temperature (Tm=93.5 °C) 6.2 °C below that of the holoenzyme (99.7 °C), identifying Zn2+ as a stabilizing feature in TNAK. TNAK is a monomeric enzyme with a molecular mass of approx. 25 kDa. TNAK displays Vmax and Km values at 30 °C identical with those of the E. coli AK at 30 °C, and displays very high activity at 80 °C, with a specific activity above 8000 units/mg. The unusually high activity of TNAK at 30 °C makes it an interesting model to test the role of enzyme flexibility in activity.