There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
MMAB (methylmalonic aciduria type B) is a mitochondrial enzyme involved in the metabolism
of vitamin B(12). It functions as the ATP:cob(I)alamin adenosyltransferase for the
generation of adenosylcobalamin (AdoCbl), the cofactor of methylmalonyl-CoA mutase
(MCM). Impaired MMAB activity leads to the inherited disorder methylmalonic aciduria
and is responsible for the cblB complementation group. In this study, the effects
on substrate binding of two catalytically inactive patient mutations, R190H and R186W,
were investigated using intrinsic fluorescence quenching of MMAB as a measure of ligand-binding.
We report the dissociation constant (K(d)) of wild-type MMAB for HOCbl is 51 microM
and for ATP is 365 microM and show that cobalamin enhances the affinity of MMAB for
ATP, while ATP does not show detectable effects on cobalamin binding. We confirm that
residue Arg190 plays a role in the formation of the ATP-binding site as described
previously [H.L. Schubert, C.P. Hill, Structure of ATP-bound human ATP:cobalamin adenosyltransferase,
Biochemistry 45 (2006) 15188-15196]. Unexpectedly, mutation R186W does not disrupt
the binding of HOCbl to MMAB as predicted; instead, both R190H and R186W significantly
disrupt the affinity between MMAB and AdoCbl. We surmise that these two residues may
be critical for the transfer of the 5'-deoxyadenosyl group from ATP to cob(I)alamin,
possibly by contributing to the precise positioning of the two substrates to permit
catalysis to occur. Characterization of ligand-binding by MMAB provides insight into
the mechanism of cobalamin adenosylation and the effect of patient mutations in the
inherited disorder.