N. Rashid et al., A unique DNase activity shares the active site with ATPase activity of theRecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon, FEBS LETTER, 445(1), 1999, pp. 111-114
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.