Loss of Allostery and Coenzyme B₁₂ Delivery by a Pathogenic Mutation in Adenosyltransferase

Abstract

ATP-dependent cob(I)alamin adenosyltransferase (ATR) is a bifunctional protein; an enzyme that catalyzes the adenosylation of cob(I)alamin and an escort that delivers the product, adenosylcobalamin (AdoCbl or coenzyme B12), to methylmalonyl-CoA mutase (MCM), resulting in holoenzyme formation. Failure to assemble holo-MCM leads to methylmalonic aciduria. We have previously demonstrated that only two equivalents of AdoCbl bind per homotrimer of ATR and that binding of ATP to the vacant active site triggers ejection of one equivalent of AdoCbl from an adjacent site. In this study, we have mimicked in the Methylobacterium extorquens ATR, a C-terminal truncation mutation, D180X, described in a patient with methylmalonic aciduria, and characterized the associated biochemical penalties. We demonstrate that while kcat and KMCob(I) for D180X ATR are only modestly decreased (by 3- and 2-fold, respectively), affinity for the product, AdoCbl, is significantly diminished (400-fold) and the negative cooperativity associated with its binding is lost. We also demonstrate that the D180X mutation corrupts ATP-dependent cofactor ejection, which leads to transfer of AdoCbl from wild-type ATR to MCM. These results suggest that the pathogenicity of the corresponding human truncation mutant results from its inability to sequester AdoCbl for direct transfer to MCM. Instead, cofactor release into solution is predicted to reduce the capacity for holo-MCM formation, leading to disease.

The C-terminal domain of CblD interacts with CblC and influences intracellular cobalamin partitioning

“…1). In the bacterial orthologs of these mitochondrial proteins, adenosyltransferase synthesizes and then directly transfer AdoCbl to the mutase·CblA complex [40-42]. In bacteria, orthologs of CblD and CblC are not found; hence the details of the cofactor docking mechanisms are likely to be different from the mammalian pathway.…”

A switch III motif relays signaling between a B12 enzyme and its G-protein chaperone

“…Recombinant MeaB, MCM, ATR, and malonyl-CoA synthetase were expressed and purified from One Shot® BL21 (DE3) chemically competent E. coli (Invitrogen) as described previously 6,34,35 . Following purification, the enzymes were stored at −80 °C in 50 mM Hepes buffer, pH 8.0, 0.3 M KCl, 10 mM MgCl 2 , 5 % glycerol (Buffer A).…”

Cofactor Editing by the G-protein Metallochaperone Domain Regulates the Radical B12 Enzyme IcmF

“…Two proteins, adenosyltransferase (ATR) (9) and a G-protein chaperone (10), are needed for cofactor repair and reloading. The Methylobacterium extorquens orthologs of MCM, ATR and the G-protein (known as MeaB), have been characterized extensively (8,(11)(12)(13)(14)(15)(16)(17), and our understanding of the mechanism of cofactor repair derives primarily from studies on this system. The importance of the chaperonedependent cofactor loading and repair mechanisms is underscored by the existence of disease-causing mutations in both human ATR and the G-protein chaperone (18,19).…”