Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: Crystallographic evidence
Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: Crystallographic evidence
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DOI：
10.1021/bi962337c
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发表时间：
1997-01-21
期刊：
影响因子：
2.9
通讯作者：
Kraut, J
中科院分区：
文献类型：
--
作者：
Sawaya, MR;Kraut, J
由大肠杆菌二氢叶酸还原酶（ecDHFR）催化的反应循环通过五个可检测的动力学中间体：全酶、米氏复合物、三元产物复合物、四氢叶酸（THF）二元复合物和THF NADPH复合物。与这五种中间体和过渡态（如甲氨蝶呤NADPH复合物所示）类似的同晶晶体结构已用于组装2.1埃分辨率的电影，描绘催化循环期间的环和子域运动（见支持信息）。结构表明，M20环主要是封闭的全酶，米氏，过渡态复合物的反应物。但是，在循环的剩余部分，当烟酰胺未结合时，环封闭（突出）烟酰胺-核糖结合口袋。当从闭合构象变为封闭构象时，环的中心部分从β-折叠重排为3（10）螺旋。这种变化可能是通过不规则结构的开环构象发生的，它可以短暂地允许水分子进入使二氢叶酸的N5质子化的位置。从Michaelis到过渡态类似物复合物，ecDHFR的两个半部分（腺苷结合亚结构域和环亚结构域）之间的旋转使（对氨基苯甲酰基）谷氨酸盐（pABG）结合裂缝闭合约0.5埃。由此产生的与pABG部分接触的增强可能会稳定过渡态蝶啶环C6处的褶皱。通过螺旋B和C的辅因子诱导的运动（约0.5埃）进一步调节亚结构域旋转，在THF NADPH类似物复合物中产生比在THF类似物复合物中更大的pABG裂缝。这样的运动可以解释如何通过NADPH结合来辅助THF释放。在脊椎动物DHFR结构中未观察到亚结构域旋转，但类似的环运动（残基59-70）似乎类似地调节pABG裂缝宽度，表明这些运动对催化很重要。在脊椎动物DHFR结构中也未观察到的环运动可能优先削弱ecDHFR中NADP（+）与NADPH的结合，这是一种进化适应，以减少原核生物富含NADP（+）环境中的产物抑制。
The reaction catalyzed by Escherichia coli dihydrofolate reductase (ecDHFR) cycles through five detectable kinetic intermediates: holoenzyme, Michaelis complex, ternary product complex, tetrahydrofolate (THF) binary complex, and THF NADPH complex. Isomorphous crystal structures analogous to these five intermediates and to the transition state (as represented by the methotrexate NADPH complex) have been used to assemble a 2.1 Angstrom resolution movie depicting loop and subdomain movements during the catalytic cycle (see Supporting Information). The structures suggest that the M20 loop is predominantly closed over the reactants in the holoenzyme, Michaelis, and transition state complexes. But, during the remainder of the cycle, when nicotinamide is not bound, the loop occludes (protrudes into) the nicotinamide-ribose binding pocket. Upon changing from the closed to the occluded conformation, the central portion of the loop rearranges from beta-sheet to 3(10) helix. The change may occur by way of an irregularly structured open loop conformation, which could transiently admit a water molecule into position to protonate N5 of dihydrofolate. From the Michaelis to the transition state analogue complex, rotation between two halves of ecDHFR, the adenosine binding subdomain and loop subdomain, closes the (p-aminobenzoyl)glutamate (pABG) binding crevice by approximate to 0.5 Angstrom. Resulting enhancement of contacts with the pABG moiety may stabilize puckering at C6 of the pteridine ring in the transition state. The subdomain rotation is further adjusted by cofactor-induced movements (approximate to 0.5 Angstrom) of helices B and C, producing a larger pABG cleft in the THF NADPH analogue complex than in the THF analogue complex. Such movements may explain how THF release is assisted by NADPH binding. Subdomain rotation is not observed in vertebrate DHFR structures, but an analogous loop movement (residues 59-70) appears to similarly adjust the pABG cleft width, suggesting that these movements are important for catalysis. Loop movement, also unobserved in vertebrate DHFR structures, may preferentially weaken NADP(+) vs NADPH binding in ecDHFR, an evolutionary adaptation to reduce product inhibition in the NADP(+) rich environment of prokaryotes.