A unique DNase activity shares the active site with ATPase activity of theRecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon

A RecA/Rad51 homologue from Pyrococcus koda-karaensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues, Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesti ng that the catalytic centers of the ATPase and deoxyribonuclease activitie s are overlapped. To examine whether these two enzymatic activities share t he same active site, a number of site-directed mutations were introduced in to Pk-REC and the ATPase and deoxyribonuclease activities of the mutant pro teins were determined. The mutant enzyme in which double mutations Lys-33 t o Ala and Thr-34 to Ala were introduced, fully lost both of these activitie s, indicating that Lys-33 and/or Thr-34 are important for both ATPase and d eoxyribonuclease activities. The mutation of Asp-112 to Ale slightly and al most equally reduced both ATPase and deoxyribonuclease activities. In addit ion, the mutation of Glu-54 to Gib did not seriously affect the ATPase, deo xyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk- REC are common, It is noted that unlike Glu-96 in Escherichia coli RecA, wh ich has been proposed to be a catalytic residue for the ATPase activity, th e corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein. (C) 1999 Federation of European Biochemical Societ ies.