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A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue ( Pk ‐REC) from a hyperthermophilic archaeon
Author(s) -
Rashid Naeem,
Morikawa Masaaki,
Kanaya Shigenori,
Atomi Haruyuki,
Imanaka Tadayuki
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)00107-6
Subject(s) - deoxyribonuclease , biochemistry , atpase , biology , deoxyribonuclease i , mutant , enzyme , escherichia coli , mutation , microbiology and biotechnology , dna , gene , base sequence
A RecA/Rad51 homologue from Pyrococcus kodakaraensis 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, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site‐directed mutations were introduced into Pk ‐REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys‐33 to Ala and Thr‐34 to Ala were introduced, fully lost both of these activities, indicating that Lys‐33 and/or Thr‐34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp‐112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu‐54 to Gln did not seriously affect the ATPase, deoxyribonuclease, 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, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu‐54 in Pk ‐REC is not involved in the catalytic function of the protein.