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Abstract

NADP-dependent methylene-tetrahydromethanopterin (methylene-H₄MPT) dehydrogenase (MtdA) catalyzes the reversible dehydrogenation of methylene-H₄MPT to form methenyl-H₄MPT⁺ by using NADP⁺ as a hydride acceptor. This hydride transfer reaction is involved in the oxidative metabolism from formaldehyde to CO₂ 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-H₄MPT 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-H₄MPT that optimizes the geometry for hydride transfer. An additional 1.5 Å structure of MtdA in complex with the nonreactive NADP⁺ and methenyl-H₄MPT⁺ 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-H₄MPT bound to MtdA is smaller than that in the enzyme-free state but is similar to that in H_2- and F₄₂₀-dependent methylene-H₄MPT 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 H₄MPT-dependent enzymes