Satapathy Ajit K, Pavankumar Theetha L, Bhattacharjya Sumana, Sankaranarayanan Rajan, Ray Malay K
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, India.
FEBS J. 2008 Apr;275(8):1835-51. doi: 10.1111/j.1742-4658.2008.06342.x. Epub 2008 Mar 9.
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.
RecD对于南极嗜冷细菌丁香假单胞菌Lz4W在低温下的生长至关重要。为了研究其活性的本质，我们分析了野生型和突变型RecD蛋白，这些蛋白在七个保守解旋酶基序中的每一个中都有重要残基的替换。野生型RecD在体外表现出依赖DNA的ATP酶和解旋酶活性，能够解开仅含有5'突出端或叉状末端的短DNA双链体。五个突变蛋白，即基序I中的K229Q、基序II中的D323N和E324Q、基序III中的Q354E以及基序VI中的R660A，完全丧失了ATP酶和解旋酶活性。其他三个突变体，基序Ia中的T259A、基序IV中的R419A和基序V中的E633Q，ATP酶活性表现出不同程度的降低，但没有解旋酶活性。虽然所有RecD蛋白都具有DNA结合活性，但基序IV和V的突变体表现出结合能力降低，而基序II突变体对单链DNA的结合程度更高。值得注意的是，只有具有体外ATP酶活性的RecD变体能够补充丁香假单胞菌recD失活菌株在4℃下的冷敏感生长。这些结果表明，低温下对RecD的需求在于其ATP水解活性。
