Structural and kinetic investigations of the carboxy terminus of NADPH-cytochrome P450 oxidoreductase.
Structural and kinetic investigations of the carboxy terminus of NADPH-cytochrome P450 oxidoreductase.
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NADPH-细胞色素P450氧化还原酶羧基末端的结构和动力学研究
DOI：
10.1016/j.abb.2021.108792
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发表时间：
2021-04-15
影响因子：
3.9
通讯作者：
Kim JP
中科院分区：
文献类型：
--
作者：
Hubbard PA;Xia C;Shen AL;Kim JP
研究了 NADPH-细胞色素 P450 氧化还原酶 (CYPOR) 的侧链和羧基末端残基的位置对催化活性、羧基末端结构以及与辅因子相互作用的影响。残基 Asp675 和 Val676 的串联删除预计会改变功能上重要的 Trp677 的位置，导致细胞色素 c 还原酶活性高于之前关于 Asp675 和 Trp677 在催化中的重要性的研究中预期的活性。该变体结构的晶体学测定揭示了羧基末端的两种构象。在一种构象 (Mol A) 中，最后一个 α 螺旋部分解开，导致 β 链 21 中所有后续残基重新定位，从 Arg671 到 Leu674（对应于野生型结构中的 Ser673 和 Val676）。这导致两个 C 端残基 Trp677 和 Ser678 保持在其野生型位置，如野生型结构中所示，Trp677 的吲哚环与 FAD 的异咯嗪环堆叠，而 Ser673 占据与催化残基 Asp675 相似的位置。另一种更无序的构象是 Mol A 构象和最后一个 α 螺旋未解开的构象的混合物，烟酰胺环处于两种构象之一，如在野生型结构 (1AMO) 中观察到的那样朝向蛋白质表面，或与黄素环堆叠，类似于缺乏 Trp677 和 Ser678 (1JA0) 的 W677X 结构中所见的情况。对其他变体的进一步动力学分析显示，Trp677 的丙氨酸或甘氨酸的删除或取代以及 Ser678 的删除，导致 CYPOR 与 NADP+、2'5'-ADP 和 2'-AMP 相互作用的改变，以及细胞色素 c 还原酶活性的 pH 依赖性的改变。我们假设删除羧基末端的大残基可以增加移动性，从而导致对 NADP+ 的 2'5'-ADP 和 2'-AMP 部分的亲和力降低以及随后的结构域移动。
The influence of the side chains and positioning of the carboxy-terminal residues of NADPH-cytochrome P450 oxidoreductase (CYPOR) on catalytic activity, structure of the carboxy terminus, and interaction with cofactors has been investigated. A tandem deletion of residues Asp675 and Val676 that was expected to shift the position of the functionally important Trp677, resulted in higher cytochrome c reductase activity than that expected from previous studies on the importance of Asp675 and Trp677 in catalysis. Crystallographic determination of the structure of this variant revealed two conformations of the carboxy terminus. In one conformation (Mol A), the last α-helix is partially unwound, resulting in repositioning of all subsequent residues in β-strand 21, from Arg671 to Leu674 (corresponding to Ser673 and Val676 in the wild type structure). This results in the two C-terminal residues, Trp677 and Ser678, being maintained in their wild type positions, with the indole ring of Trp677 stacked against the isoalloxazine ring of FAD as seen in the wild type structure, and Ser673 occupying a similar position to the catalytic residue, Asp675. The other, more disordered conformation is a mixture of the Mol A conformation and one in which the last α-helix is not unwound and the nicotinamide ring is in one of two conformations, out towards the protein surface as observed in the wild type structure (1AMO), or stacked against the flavin ring, similar to that seen in the W677X structure that lacks Trp677 and Ser678 (1JA0). Further kinetic analysis on additional variants showed deletion or substitution of alanine or glycine for Trp677 in conjunction with deletion of Ser678 produced alterations in interactions of CYPOR with NADP+, 2’5’-ADP, and 2’-AMP, as well as the pH dependence of cytochrome c reductase activity. We postulate that deletion of bulky residues at the carboxy terminus permits increased mobility leading to decreased affinity for the 2’5’-ADP and 2’-AMP moieties of NADP+ and subsequent domain movement.
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通讯作者：
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影响因子：
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