Abstract
Vitamin B(12) (cobalamin, Cbl) is essential to the function of two human enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MUT). The conversion of dietary Cbl to its cofactor forms, methyl-Cbl (MeCbl) for MS and adenosyl-Cbl (AdoCbl) for MUT, located in the cytosol and mitochondria, respectively, requires a complex pathway of intracellular processing and trafficking. One of the processing proteins, MMAA (methylmalonic aciduria type A), is implicated in the mitochondrial assembly of AdoCbl into MUT and is defective in children from the cblA complementation group of cobalamin disorders. To characterize the functional interplay between MMAA and MUT, we have crystallized human MMAA in the GDP-bound form and human MUT in the apo, holo, and substrate-bound ternary forms. Structures of both proteins reveal highly conserved domain architecture and catalytic machinery for ligand binding, yet they show substantially different dimeric assembly and interaction, compared with their bacterial counterparts. We show that MMAA exhibits GTPase activity that is modulated by MUT and that the two proteins interact in vitro and in vivo. Formation of a stable MMAA-MUT complex is nucleotide-selective for MMAA (GMPPNP over GDP) and apoenzyme-dependent for MUT. The physiological importance of this interaction is highlighted by a recently identified homoallelic patient mutation of MMAA, G188R, which, we show, retains basal GTPase activity but has abrogated interaction. Together, our data point to a gatekeeping role for MMAA by favoring complex formation with MUT apoenzyme for AdoCbl assembly and releasing the AdoCbl-loaded holoenzyme from the complex, in a GTP-dependent manner.
Highlights
In humans, vitamin B12 is the cofactor for two enzymes: cytosolic methionine synthase and mitochondrial methylmalonyl-CoA mutase (MUT,3 known as MCM), utilizing methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) as cofactor forms, respectively
A band corresponding to human MUT (hMUT) was detected in cells expressing human MMAA (hMMAA)-Flag, but not in Flag-only cells or cells expressing hMMAAG188R-Flag. These data demonstrate that hMMAA and hMUT form a complex in vivo and in vitro, which is abolished with the G188R mutation in hMMAA
We have examined the structures of hMUT and hMMAA, demonstrating a nucleotide-dependent interaction of the two proteins and a gating behavior of hMMAA toward cobalamin binding of hMUT, and confirm the occurrence of a protein complex in cells in vivo but not in a GTPase-active mutant protein carrying the G188R substitution
Summary
The different structural configuration of the human enzyme. Unlike psMUT, which is a heterodimer of one catalytic (␣) and one acatalytic subunit () [13], hMUT is a homodimer with two catalytic (␣) subunits per dimer [14]. A role for MeaB has been recently proposed in gating AdoCbl transfer to MUT in order to discriminate against the binding of inactive Cbl forms, in a manner dependent upon GTP binding and hydrolysis [20]. These results suggest that the human proteins hMMAA and hMUT might functionally interact in a similar manner. In this study we determine, by x-ray crystallography, the structures of hMMAA and hMUT We show that both proteins are homodimeric in native state albeit with different modes of dimeric assembly compared with their bacterial counterparts. We show that complex formation and GTPase stimulation are abrogated in hMMAA with the G188R patient mutation
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