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@article{Lyatuu_Parthasarathy_Zhou_Zelenka_Zelder_Buckel_2021, title={Probing the cob(II)alamin Cond UctorHhypothesis with Glutamate Mutase from Clostridium Cochlearium}, volume={38}, url={https://tjs.udsm.ac.tz/index.php/tjs/article/view/439}, abstractNote={<p>It had been proposed that during reversible coenzyme B<sub>12</sub> 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 C<sub>19</sub>-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<sup>-1</sup>. We tested this hypothesis with glutamate mutase and artificial coenzyme B<sub>12</sub> derivatives. The assembly of coenzyme B<sub>12</sub> (adenosylcobalamin) with recombinant components GlmS and GlmE of glutamate mutase from <em>Clostridium cochlearium</em> 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 B<sub>12</sub> and 3',5'- dideoxyadenosylcobalamin suggested similar binding properties of the cofactors to the apoenzyme. However, the catalytic efficiency (k<sub>cat</sub>/K<sub>m</sub>) of glutamate mutase was 15 times reduced with 3',5'-dideoxyadenosylcobalamin compared to catalysis with coenzyme B<sub>12</sub> as cofactor. This translates into a stabilization of only 7 kJ mol<sup>-1</sup> in the substrate activation step. We attribute this effect to weak interactions of O3' of the riboise moiety with either C<sub>19</sub>-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.</p>}, number={3}, journal={Tanzania Journal of Science}, author={Lyatuu, FE and Parthasarathy, A and Zhou, K and Zelenka, K and Zelder, FH and Buckel, W}, year={2021}, month={Apr.}, pages={148–156} }