Smith Nathan, Horswill Alexander R, Wilson Mark A
Department of Biochemistry and Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE, 68588.
Department of Immunology & Microbiology, University of Colorado Anschutz School of Medicine, Aurora, CO 80045.
bioRxiv. 2023 Nov 8:2023.11.07.566054. doi: 10.1101/2023.11.07.566054.
Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate. Bacterial DHFRs are targets of several important antibiotics as well as model enzymes for the role of protein conformational dynamics in enzyme catalysis. We collected 0.93 Å resolution X-ray diffraction data from both (Bs) and (Ec) DHFRs bound to folate and NADP. These oxidized ternary complexes should not be able to perform chemistry, however electron density maps suggest hydride transfer is occurring in both enzymes. Comparison of low- and high-dose EcDHFR datasets show that X-rays drive partial production of tetrahydrofolate. Hydride transfer causes the nicotinamide moiety of NADP to move towards the folate as well as correlated shifts in nearby residues. Higher radiation dose also changes the conformational heterogeneity of Met20 in EcDHFR, supporting a solvent gating role during catalysis. BsDHFR has a different pattern of conformational heterogeneity and an unexpected disulfide bond, illustrating important differences between bacterial DHFRs. This work demonstrates that X-rays can drive hydride transfer similar to the native DHFR reaction and that X-ray photoreduction can be used to interrogate catalytically relevant enzyme dynamics in favorable cases.
二氢叶酸还原酶（DHFR）催化二氢叶酸依赖NADPH还原为四氢叶酸。细菌DHFR是几种重要抗生素的作用靶点，也是蛋白质构象动力学在酶催化中作用的模型酶。我们收集了结合叶酸和NADP的嗜热栖热放线菌（Bs）和大肠杆菌（Ec）DHFR的分辨率为0.93 Å的X射线衍射数据。这些氧化的三元复合物应该无法进行化学反应，然而电子密度图表明两种酶中都发生了氢化物转移。低剂量和高剂量EcDHFR数据集的比较表明，X射线驱动了四氢叶酸的部分生成。氢化物转移导致NADP的烟酰胺部分向叶酸移动以及附近残基的相关位移。更高的辐射剂量也改变了EcDHFR中Met20的构象异质性，支持了催化过程中的溶剂门控作用。BsDHFR具有不同的构象异质性模式和一个意外的二硫键，说明了细菌DHFR之间的重要差异。这项工作表明，X射线可以驱动类似于天然DHFR反应的氢化物转移，并且在有利的情况下，X射线光还原可用于探究与催化相关的酶动力学。
