H2-Forming N5,N10-Methylenetetrahydromethanopterin Dehydrogenase from Methanobacterium thermoautotrophicum Catalyzes a Stereoselective Hydride Transfer As Determined by Two-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, N10-methenyl-5,6,7,8-tetrahydromethanopterin at C(14a) with H2 to N5, N10-methylene-5,6,7,8-tetrahydromethanopterin can be catalyzed by H2-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 H2 is transferred by the enzyme into the rel-(pro-R) position of the C(14a) methylene group of the reaction product N5, N10-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 degree C and pH 6.5).(ABSTRACT TRUNCATED AT 250 WORDS)