Assembly and protection of the radical enzyme, methylmalonyl-CoA mutase, by its chaperone.
MeaB is a recently described P-loop GTPase that plays an auxiliary role in the reaction catalyzed by the radical B12 enzyme, methylmalonyl-CoA mutase. Defects in the human homologue of MeaB result in methylmalonic aciduria, but the role of this protein in coenzyme B12 assimilation and/or utilization is not known. Methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA that uses reactive radical intermediates that are susceptible to oxidative inactivation. In this study, we have examined the influence of MeaB on the kinetics of the reaction catalyzed by methylmalonyl-CoA mutase and on the thermodynamics of cofactor binding. MeaB alone has a modest effect on the affinity of the mutase for the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor, increasing it 2-fold from 404 +/- 71 to 210 +/- 22 nM. However, in the presence of GDP, the affinity for the cofactor decreases 5-fold to 1.89 +/- 0.33 microM, while in the presence of guanosine 5'(beta-gamma imino)triphosphate, a nonhydrolyzable analogue of GTP, the binding of AdoCbl to the mutase is not detected. Protection against oxidative inactivation of the mutase by MeaB is dependent upon the presence of nucleotides with the MeaB/GDP and MeaB/GTP complexes decelerating the rate of formation of oxidized cofactor by 3- and 15-fold, respectively. This study suggests that MeaB functions in the GTP-dependent assembly of holomethylmalonyl-CoA mutase and subsequent protection of radical intermediates during catalysis.
Padovani
Dominique
D
Redox Biology Center and Biochemistry Department, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Banerjee
Ruma
R
D002151:Calorimetry; D003038:Cobamides / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008765:Methylmalonyl-CoA Mutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000235:genetics
D003038:Cobamides; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D020558:GTP Phosphohydrolases; D008765:Methylmalonyl-CoA Mutase; C000913:cobamamide