TAILIEUCHUNG - Báo cáo khoa học: ATPase activity of RecD is essential for growth of the Antarctic Pseudomonas syringae Lz4W at low temperature

RecD is essential for growth at low temperature in the Antarctic psychro-trophic bacterium Pseudomonas syringaeLz4W. To examine the essential nature of its activity, we analyzed wild-type and mutant RecD proteins with substitutions of important residues in each of the seven conserved helicase motifs. | ỊFEBS Journal ATPase activity of RecD is essential for growth of the Antarctic Pseudomonas syringae Lz4W at low temperature Ajit K. Satapathy Theetha L. Pavankumar Sumana Bhattacharjya Rajan Sankaranarayanan and Malay K. Ray Centre for Cellular and Molecular Biology Hyderabad India Keywords cold adaptation Pseudomonas syringae RecBCD enzyme RecD ATPase RecD helicase Correspondence M. K. Ray Centre for Cellular and Molecular Biology UppalRoad Hyderabad 500007 India Fax 91 40 2716 0591 Tel 91 40 2719 2512 E-mail malay@ Received 10 October 2007 revised 12 February 2008 accepted 18 February 2008 doi RecD is essential for growth at low temperature in the Antarctic psychro-trophic bacterium Pseudomonas syringae Lz4W. To examine the essential nature of its activity we analyzed wild-type and mutant RecD proteins with substitutions of important residues in each of the seven conserved helicase motifs. The wild-type RecD displayed DNA-dependent ATPase and helicase activity in vitro with the ability to unwind short DNA duplexes containing only 5 overhangs or forked ends. Five of the mutant proteins K229Q in motif I D323N and E324Q in motif II Q354E in motif III and R660A in motif VI completely lost both ATPase and helicase activities. Three other mutants T259A in motif Ia R419A in motif IV and E633Q in motif V exhibited various degrees of reduction in ATPase activity but had no helicase activity. While all RecD proteins had DNA-binding activity the mutants of motifs IV and V displayed reduced binding and the motif II mutant showed a higher degree of binding to ssDNA. Significantly only RecD variants with in vitro ATPase activity could complement the cold-sensitive growth of a recD-inactivated strain of P. syringae at 4 C. These results suggest that the requirement for RecD at lower temperatures lies in its ATP-hydrolyzing activity. RecD is a 5 fi 3 helicase motor protein. The primary sequence contains the characteristic seven .

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