The Hydride Transfer Process in NADP-dependent Methylene-tetrahydromethanopterin Dehydrogenase.

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.