The crystal structure of a tetrahydrofolate-bound dihydrofolate reductase reveals the origin of slow product release
The crystal structure of a tetrahydrofolate-bound dihydrofolate reductase reveals the origin of slow product release
复制标题
DOI：
10.1038/s42003-018-0236-y
复制
发表时间：
2018-01-01
影响因子：
5.9
通讯作者：
Skolnick, Jeffrey
中科院分区：
文献类型：
--
作者：
Cao, Hongnan;Gao, Mu;Skolnick, Jeffrey
二氢叶酸还原酶（DHFR）催化7，8-二氢叶酸（FH 2）通过氢化物转移从NADPH立体特异性还原为（6s）-5，6，7，8-四氢叶酸（FH 4）。共识大肠杆菌DHFR机制涉及在限速产物释放步骤期间发生的闭合和闭塞状态之间的构象变化。虽然蛋白质数据库（PDB）包含超过250个DHFR结构，但负责限速产物释放的FH 4复合物结构是未知的。我们报告我们所知的第一个晶体结构的E。杆菌DHFR：FH 4复合物在1.03埃分辨率下显示出在FH 2或相关的（6 R）-5，10-二脱氮-FH 4复合物中不存在的明显的稳定化相互作用。我们发现在晶体生长过程中共纯化的内源性FH 4衰变的时间过程，从FH 4到FH 2的转化发生在2-3天内。我们还确定了E.大肠杆菌DHFR与缓慢起效的纳摩尔抑制剂，与氨甲喋呤复合物形成对比，提出了一个合理的策略，设计DHFR抗生素的目标FH 4产品构象。
Dihydrofolate reductase (DHFR) catalyzes the stereospecific reduction of 7,8-dihydrofolate (FH2) to (6s)-5,6,7,8-tetrahydrofolate (FH4) via hydride transfer from NADPH. The consensus Escherichia coli DHFR mechanism involves conformational changes between closed and occluded states occurring during the rate-limiting product release step. Although the Protein Data Bank (PDB) contains over 250 DHFR structures, the FH4 complex structure responsible for rate-limiting product release is unknown. We report to our knowledge the first crystal structure of an E. coli. DHFR: FH4 complex at 1.03 angstrom resolution showing distinct stabilizing interactions absent in FH2 or related (6R)-5,10-dideaza-FH4 complexes. We discover the time course of decay of the co-purified endogenous FH4 during crystal growth, with conversion from FH4 to FH2 occurring in 2-3 days. We also determine another occluded complex structure of E. coli DHFR with a slow-onset nanomolar inhibitor that contrasts with the methotrexate complex, suggesting a plausible strategy for designing DHFR antibiotics by targeting FH4 product conformations.