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Schlagwörter:
conformational changes; dehydrogenase; enzyme catalysis; one-carbon metabolism; X-ray crystallography
Zusammenfassung:
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 H2- 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
