Thermotoga neapolitana adenylate kinase is highly active at 30°C

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 fitration data indicate a content of 1 mol of Zn2+/mol of recombinant TNAK. The EDTA-treated enzyme has a melting temperature (T-m = 93.5 degreesC) 6.2 degreesC below that of the holoenzyme (99.7 degreesC), identifying Zn2+ as a stabilizing feature in TNAK. TNAK is a monomeric enzyme with a molecular mass of approx. 25 kDa. TNAK displays V-max and K-m values at 30 degreesC identical with those of the E. coli AK at 30 degreesC, and displays very high activity at 80 degreesC, with a specific activity above 8000 units/mg. The unusually high activity of TNAK at 30 degreesC makes it an interesting model to test the role of enzyme flexibility in activity.