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首页> 外文期刊>Biochemistry >Novel insights into catalytic mechanism from a crystal structure of human topoisomerase I in complex with DNA.
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Novel insights into catalytic mechanism from a crystal structure of human topoisomerase I in complex with DNA.

机译：与DNA复合物中人型甲基异构酶I晶体结构催化机理的新洞察。

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Human topoisomerase I helps to control the level of DNA supercoiling in cells and is vital for numerous DNA metabolic events, including replication, transcription, and recombination. The 2.6 A crystal structure of human topoisomerase I in noncovalent complex with a DNA duplex containing a cytosine at the -1 position of the scissile strand rather than the favored thymine is reported. The hydrogen bond between the O2 position of this -1 base and the epsilon-amino of the conserved Lys-532 residue, the only base-specific contact observed previously in the human topoisomerase I-DNA interaction, is maintained in this complex. Several unique features of this structure, however, have implications for the DNA-binding and active-site mechanisms of the enzyme. First, the ends of the DNA duplex were observed to shift by up to 5.4 A perpendicular to the DNA helical axis relative to structures reported previously, suggesting a novel degree of plasticity in the interaction between human topoisomerase I and its DNA substrate. Second, 12 additional residues at the NH(2) terminus of the protein (Trp-203-Gly-214) could be built in this structure, and they were found to pack against the putative hinge region implicated in the clamping of the enzyme around duplex DNA. Third, a water molecule was observed adjacent to the scissile phosphate and the active-site residues; the potential specific base character of this solvent molecule in the active-site mechanism of the enzyme is discussed. Fourth, the scissile phosphate group was found to be rotated by 75 degrees, bringing Lys-532 into hydrogen-bonding distance of one of the nonbridging phosphate oxygens. This orientation of the scissile phosphate group implicates Lys-532 as a fifth active-site residue, and also mimics the orientation observed for the 3'-phosphotyrosine linkage in the covalent human topoisomerase I-DNA complex structure. The implications of these structural features for the mechanism of the enzyme are discussed, including the potential requirement for a rotation of the scissile phosphate group during DNA strand cleavage and covalent attachment.
机译：人拓扑异构酶I有助于控制细胞中的DNA超咖啡水平，并且对于许多DNA代谢事件至关重要，包括复制，转录和重组。据报道，2.6在非共价络合物中的2.6晶体结构，在非共价络合物中，据报道含有在剪发链的-1位置的胞嘧啶的DNA双链体而不是受青胸腺嘧啶。在该复合物中，将在本综合体中保持在人拓扑异构酶I-DNA相互作用中观察到的-1碱基和ε-氨基之间的氢键与保守的Lys-532残基的ε-氨基之间的氢键。然而，这种结构的几种独特特征对酶的DNA结合和有效点机制具有影响。首先，观察到DNA双链体的末端相对于先前报道的结构垂直于5.4垂直于DNA螺旋轴线，表明人型拓扑异构酶I及其DNA底物之间的相互作用具有新的可塑性。其次，在该结构中可以建立蛋白质（TRP-203-GLY-214）的NH（2）末端的12个另外的残基，并且发现它们抵靠涉及酶夹紧的推定铰链区域包装双相DNA。第三，观察到漂移磷酸盐和活性位残留物附近的水分子;讨论了该酶的溶剂分子中该溶剂分子的潜在特异性碱性碱性。第四，发现股次磷酸酯基团将旋转75度旋转，使Lys-532导致磷酸氧化氧基之一的氢键结合距离。漂移磷酸基团的这种取向意味着Lys-532作为第五个活性位点残留物，并且还模仿在共价人拓扑异构酶I-DNA复合结构中为3'-磷酸酪氨酸连杆观察到的取向。讨论了这些结构特征对酶机制的影响，包括在DNA链裂解和共价附着期间旋转磷酸酯基团旋转的潜在要求。

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