H-2-FORMING N-5,N-10-METHYLENETETRAHYDROMETHANOPTERIN DEHYDROGENASE FROM METHANOBACTERIUM-THERMOAUTOTROPHICUM CATALYZES A STEREOSELECTIVE HYDRIDE TRANSFER AS DETERMINED BY 2-DIMENSIONAL NMR-SPECTROSCOPY

5,6,7,8-Tetrahydromethanopterin is a coenzyme playing a key role in the energy metabolism of methanogenic archaea. In Methanobacterium thermoautotrophicum, the reduction of N5,N-10-methenyl-5,6,7,8-tetrahydromethanopterin at C(14a) with H-2 to N5,N-10-methylene-5,6,7,8-tetrahydromethanopterin can be catalyzed by H-2-forming methylenetetrahydromethanopterin dehydrogenase, a new hydrogenase present in most methanogenic archaea, which is unique because it does not contain nickel or iron/sulfur clusters. In this work, the stereochemistry of this enzymatic hydride-transfer reaction is elucidated by means of a series of heteronuclear two-dimensional NMR experiments. It is found that the hydride from H-2 is transferred by the enzyme into the rel-(pro-R) position of the C(14a) methylene group of the reaction product N5,N-10-methylene-5,6,7,8-tetrahydromethanopterin. NMR experiments are described that show that the hydrogen nucleus of the hydride transferred to the oxidized coenzyme partially originates from water. The stereochemical course of this reaction is the same as that for direct hydride transfer. It is demonstrated that the diastereotopic atoms at C(14a) of the reaction product epimerize in an uncatalyzed reaction under the conditions of operation of the enzyme (k = 0.01 s-1 at 58-degrees-C and pH 6.5). On the basis of the known relative configuration of the pterin moiety of 5,6,7,8-tetrahydromethanopterin [Schleucher, J., Schworer, B., Zirngibl, C., Koch, U., Weber, W., Egert, E., Thauer, R. K., & Griesinger, C. (1992) FEBS Lett. 314, 440-444], the absolute configuration of this moiety is tentatively assigned to be (6S,7S, 11R) on the basis of a comparison of the CD spectra of N5,N-10-methenyl-5,6,7,8-tetrahydromethanopterin and its analog N5,N-10-methenyl-5,6,7,8-tetrahydrofolate. Given this absolute configuration of the pterin moiety, the rel-(pro-R) stereochemistry of the C(14a) methylene proton corresponds to the absolute (pro-R) stereochemistry.