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 psychrotrophic 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 degrees C. These results suggest that the requirement for RecD at lower temperatures lies in its ATP-hydrolyzing activity.
Satapathy
Ajit K
AK
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, India.
Pavankumar
Theetha L
TL
Bhattacharjya
Sumana
S
Sankaranarayanan
Rajan
R
Ray
Malay K
MK
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000864:Antarctic Regions; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D043211:Exodeoxyribonuclease V / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D050296:Microbial Viability; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044224:Pseudomonas syringae / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature
D006639:Histidine; D004247:DNA; D043211:Exodeoxyribonuclease V; D000251:Adenosine Triphosphatases