Site-directed mutagenesis reveals roles for conserved amino acid residues in the hexameric DNA helicase DnaB from Bacillus stearothermophilus
Site-directed mutagenesis reveals roles for conserved amino acid residues in the hexameric DNA helicase DnaB from Bacillus stearothermophilus
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DOI：
10.1093/nar/gkf527
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发表时间：
2002-09-15
影响因子：
14.9
通讯作者：
Wigley, DB
中科院分区：
文献类型：
--
作者：
Soultanas, P;Wigley, DB
对嗜热脂肪芽孢杆菌（Bacillus stearothermophilus）的六聚体复制解旋酶DnaB的基序H1、H1 a、H2和H3内的保守氨基酸残基进行定点突变研究，揭示了与这些残基相关的特定功能。特别地，在活性位点（T217和D320）中与结合的Mg 2+配位的残基对于酶的功能是重要的，但对于稳定的六聚体的形成不是必需的。基序H1 a中的保守谷氨酸（E241）可能参与水分子的活化，用于在催化过程中对结合核苷酸的γ-磷酸进行在线攻击。基序H3中保守的谷氨酰胺（Q362）作为γ-磷酸传感器，介导核苷酸和DNA结合位点的构象偶联。在这个位置上的残基的性质也是重要的引物介导的激活DnaB，这表明引物使用相同的构象偶联途径，以诱导其对DnaB的活性的刺激作用。总之，这些突变揭示了单体和六聚体解旋酶活性位点中生物化学活性的许多方面的保守性。
Site-directed mutagenesis studies on conserved amino acid residues within motifs H1, H1a, H2 and H3 of the hexameric replicative helicase DnaB from Bacillus stearothermophilus revealed specific functions associated with these residues. In particular, residues that coordinate a bound Mg2+ in the active site (T217 and D320) are important for the function of the enzyme but are not required for the formation of stable hexamers. A conserved glutamic acid (E241) in motif H1a is likely to be involved in the activation of a water molecule for in line attack on the gamma-phosphate of the bound nucleotide during catalysis. A conserved glutamine (Q362) in motif H3 acts as a gamma-phosphate sensor and mediates the conformational coupling of nucleotide- and DNA-binding sites. The nature of the residue at this position is also important for the primase-mediated activation of DnaB, suggesting that primase uses the same conformational coupling pathway to induce its stimulatory effect on the activity of DnaB. Together, these mutations reveal a conservation of many aspects of biochemical activity in the active sites of monomeric and hexameric helicases.