Crystal structure of a type II dihydrofolate reductase catalytic ternary complex
Crystal structure of a type II dihydrofolate reductase catalytic ternary complex
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DOI：
10.1021/bi701532r
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发表时间：
2007-12-25
期刊：
影响因子：
2.9
通讯作者：
London, Robert E.
中科院分区：
文献类型：
--
作者：
Krahn, Joseph M.;Jackson, Michael R.;London, Robert E.
11型二氢叶酸还原酶（DHFR）是一种质粒编码酶，可使其对细菌DHFR靶向抗叶酸药物产生耐药性。它形成一个对称的四聚体，中心孔作为活性位点。其不寻常的结构导致其混杂结合表面可容纳二氢叶酸（DHF）底物或NADPH辅助因子，这对了解其底物特异性和反应机制的努力构成了重大限制。本文描述了三元R67 DHFR中心点DHF中心点NADP（+）催化配合物的第一个结构，分辨率为1.26埃。这种结构提供了这种酶如何能够发挥作用的第一张清晰的图片，这种酶缺乏在I型DHFRs中普遍存在的活性位点羧基残基。在催化配合物中，两个对称相关的168个残基的极性主链原子提供识别基序，与烟酰胺环上的羧基酰胺和蝶啶环上的N3-O4酰胺相互作用。这组相互作用使底物和辅因子的芳香环定向在一个相对的内腔几何结构中，其中反应中心保持在很近的距离。此外，由两对Y69-Q67-Q67‘-Y69’残基组成的中心氢键网络提供了一个异常紧密的界面，这似乎可以作为一个“分子钳”，将底物固定在有利于氢化物转移的方向上。除了提供关于这种极其不寻常的酶如何能够发挥作用的第一个清晰的见解之外，三元复合物的结构还提供了关于突变挑战酶（即一种进化仅限于一次四个残基活性位点突变的酶）如何克服这一基本限制的一般见解。
Type 11 dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR center dot DHF center dot NADP(+) catalytic complex, resolved to 1.26 angstrom. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related 168 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine ring. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67'-Y69' residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationally challenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation.