Probing the cob(II)alamin Cond UctorHhypothesis with Glutamate Mutase from Clostridium Cochlearium

F. E. Lyatuu, A. Parthasarathy, K. Zhou, K. Zelenka, F. H. Zelder, W. Buckel


It had been proposed that during reversible coenzyme B12 dependent rearrangements, cob(II)alamin is not merely present as a spectator but also acts as a conductor by stabilizing the methylene radical intermediates. Density functional theory (DFT) calculations suggested a hydrogen bond between C19-H of the corrin ring and the 3'-OH moiety (O3RL) of the 5'- deoxyadenosyl radical resulting in a decrease of the activation energy by about 30 kJ mol-1. We tested this hypothesis with glutamate mutase and artificial coenzyme B12 derivatives. The assembly of coenzyme B12 (adenosylcobalamin) with recombinant components GlmS and GlmE of glutamate mutase from Clostridium cochlearium reconstitutes an active holoenzyme that catalyses the reversible rearrangement between (S)-glutamate and (2S,3S)-3-methylaspartate. Glutamate mutase activity was also demonstrated upon incubation of GlmS and E with 3',5'-dideoxyadenosylcobalamin, but not with 2',5'-dideoxyadenosylcobalamin and peptidoadenylcobalamin. In the latter cobalamin, the ribose unit of the upper ligand was replaced with a peptide mimic that contains the same number of atoms between Co(III) and the adenosine base. Measurements of the kinetic constants of glutamate mutase with coenzyme B12 and 3',5'- dideoxyadenosylcobalamin suggested similar binding properties of the cofactors to the apoenzyme. However, the catalytic efficiency (kcat/Km) of glutamate mutase was 15 times reduced with 3',5'-dideoxyadenosylcobalamin compared to catalysis with coenzyme B12 as cofactor. This translates into a stabilization of only 7 kJ mol-1 in the substrate activation step. We attribute this effect to weak interactions of O3' of the riboise moiety with either C19-H of the corrin ring or with the glutamate residue 330 of component E (Glu330). The catalytic inactivity of 2',5'- dideoxyadenosylcobalamin and peptidoadenylcobalamin reveals critical interactions of the 2'-OH moiety (O2') during the catalytic cycle. Evidence for H-bonding between O2' and Glu330 is obtained from the crystal structure analysis of glutamate mutase’s active site.

Keywords: Methylene radical; carbon skeleton rearrangement; cob(II)alamin; glutamate mutase; dideoxyadenosylcobalamin

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