Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch Straße 10, 35043, Marburg, Germany.
Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch Straße 10, 35043, Marburg, Germany; Max-Planck-Institut für Marine Mikrobiologie, Celsiusstraße 1, 28359, Bremen, Germany.
J Mol Biol. 2020 Mar 27;432(7):2042-2054. doi: 10.1016/j.jmb.2020.01.042. Epub 2020 Feb 13.
NADP-dependent methylene-tetrahydromethanopterin (methylene-HMPT) dehydrogenase (MtdA) catalyzes the reversible dehydrogenation of methylene-HMPT to form methenyl-HMPT by using NADP as a hydride acceptor. This hydride transfer reaction is involved in the oxidative metabolism from formaldehyde to CO in methylotrophic and methanotrophic bacteria. Here, we report on the crystal structures of the ternary MtdA-substrate complexes from Methylorubrum extorquens AM1 obtained in open and closed forms. Their conversion is accomplished by opening/closing the active site cleft via a 15° rotation of the NADP, relative to the pterin domain. The 1.08 Å structure of the closed and active enzyme-NADP-methylene-HMPT complex allows a detailed geometric analysis of the bulky substrates and a precise prediction of the hydride trajectory. Upon domain closure, the bulky substrate rings become compressed resulting in a tilt of the imidazolidine group of methylene-HMPT that optimizes the geometry for hydride transfer. An additional 1.5 Å structure of MtdA in complex with the nonreactive NADP and methenyl-HMPT revealed an extremely short distance between nicotinamide-C4 and imidazoline-C14a of 2.5 Å, which demonstrates the strong pressure imposed. The pterin-imidazolidine-phenyl butterfly angle of methylene-HMPT bound to MtdA is smaller than that in the enzyme-free state but is similar to that in H- and F-dependent methylene-HMPT dehydrogenases. The concept of compression-driven hydride transfer including quantum mechanical hydrogen tunneling effects, which are established for flavin- and NADP-dependent enzymes, can be expanded to hydride-transferring HMPT-dependent enzymes.
NADP 依赖性亚甲基四氢叶酸 (methylene-HMPT) 脱氢酶 (MtdA) 以 NADP 作为氢供体，催化亚甲基-HMPT 的可逆脱氢反应，生成甲烯基-HMPT。该氢转移反应参与了甲基营养菌和甲烷营养菌中甲醛到 CO 的氧化代谢。在此，我们报道了从甲基盐单胞菌 AM1 获得的开放和闭合形式的三元 MtdA-底物复合物的晶体结构。它们的转换是通过 NADP 相对于蝶呤结构域的 15°旋转，打开/关闭活性位点裂隙来完成的。闭合和活性酶-NADP-亚甲基-HMPT 复合物的 1.08 Å 结构允许对庞大的底物进行详细的几何分析，并对氢轨迹进行精确预测。在结构域关闭时，庞大的底物环被压缩，导致亚甲基-HMPT 的咪唑烷环倾斜，从而优化了氢转移的几何形状。MtdA 与非反应性的 NADP 和甲烯基-HMPT 形成复合物的另外 1.5 Å 结构揭示了烟酰胺-C4 和咪唑啉-C14a 之间的极短距离为 2.5 Å，这证明了所施加的强大压力。与游离酶相比，结合到 MtdA 的亚甲基-HMPT 的蝶呤-咪唑烷-苯蝶形角度较小，但与 H 和 F 依赖性亚甲基-HMPT 脱氢酶相似。包括量子力学氢隧穿效应的压缩驱动氢转移概念，可以扩展到氢转移的 HMPT 依赖性酶。
