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 th e energy metabolism of methanogenic archaea. In Methanobacterium therm oautotrophicum, the reduction of N5,N-10-methenyl-5,6,7,8-tetrahydrome thanopterin at C(14a) with H-2 to N5,N-10-methylene-5,6,7,8-tetrahydro methanopterin can be catalyzed by H-2-forming methylenetetrahydrometha nopterin dehydrogenase, a new hydrogenase present in most methanogenic archaea, which is unique because it does not contain nickel or iron/s ulfur clusters. In this work, the stereochemistry of this enzymatic hy dride-transfer reaction is elucidated by means of a series of heteronu clear two-dimensional NMR experiments. It is found that the hydride fr om H-2 is transferred by the enzyme into the rel-(pro-R) position of t he 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 p roduct epimerize in an uncatalyzed reaction under the conditions of op eration of the enzyme (k = 0.01 s-1 at 58-degrees-C and pH 6.5). On th e 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 moi ety is tentatively assigned to be (6S,7S, 11R) on the basis of a compa rison of the CD spectra of N5,N-10-methenyl-5,6,7,8-tetrahydromethanop terin and its analog N5,N-10-methenyl-5,6,7,8-tetrahydrofolate. Given this absolute configuration of the pterin moiety, the rel-(pro-R) ster eochemistry of the C(14a) methylene proton corresponds to the absolute (pro-R) stereochemistry.