Functional Characterization and Categorization of Missense Mutations that Cause Methylmalonyl‐ C o A Mutase ( MUT ) Deficiency

Abstract

Methylmalonyl‐CoA mutase (MUT) is an essential enzyme in propionate catabolism that requires adenosylcobalamin as a cofactor. Almost 250 inherited mutations in the MUT gene are known to cause the devastating disorder methylmalonic aciduria; however, the mechanism of dysfunction of these mutations, more than half of which are missense changes, has not been thoroughly investigated. Here, we examined 23 patient missense mutations covering a spectrum of exonic/structural regions, clinical phenotypes, and ethnic populations in order to determine their influence on protein stability, using two recombinant expression systems and a thermostability assay, and enzymatic function by measuring MUT activity and affinity for its cofactor and substrate. Our data stratify MUT missense mutations into categories of biochemical defects, including (1) reduced protein level due to misfolding, (2) increased thermolability, (3) impaired enzyme activity, and (4) reduced cofactor response in substrate turnover. We further demonstrate the stabilization of wild‐type and thermolabile mutants by chemical chaperones in vitro and in bacterial cells. This in‐depth mutation study illustrates the tools available for MUT enzyme characterization, guides future categorization of further missense mutations, and supports the development of alternative, chaperone‐based therapy for patients not responding to current treatment.

Protein destabilization and loss of protein‐protein interaction are fundamental mechanisms in cblA ‐type methylmalonic aciduria

“…This outcome is supported by the high proportion of truncating mutations and the loss of either protein stability or the ability to functionally interact with MUT in the case of missense mutations. These latter “loss of interaction” mutations are increasingly being found in the intracellular B 12 metabolic pathway, having already been identified in MMADHC, MMACHC (Froese et al., ) and MUT (Forny et al., ). This further points to complexation as required for proper B 12 pathway function, and future studies will clarify the residues required for physical and functional interaction, as well as how MMAB, and perhaps MCEE (methylmalonyl‐CoA epimerase), fit into this supramolecular multi‐protein machinery.…”

“…(). Small‐scale purification of wt MMAA and all mutants was performed as described in Forny, Froese, Suormala, Yue, and Baumgartner (). For large‐scale purification, cells were grown in a total of 12 l, then harvested by centrifugation at 5,000× g for 10 min and lysed with a Badelin Sonoplus HD2070 homogenizer.…”

“…Western Blot was performed on fresh or frozen cell pellets essentially as described in Forny et al. (), with the addition of the monoclonal anti‐flag M2 (1:2,000; Sigma–Aldrich) antibody used to detect MMAA‐flag.…”

Vitamin B12, folate, and the methionine remethylation cycle—biochemistry, pathways, and regulation

“…The MUT protein consists of two domains, an N‐terminal substrate‐binding and a C‐terminal cofactor‐binding domain connected by a short linker, which exists in a homodimeric state . Many of the patient mutations which cause the generally more severe mut 0 subtype of disease occur within the substrate‐binding domain, whereas mutations within the cofactor‐binding domain often result in the usually later onset mut − disease subtype which at least in vitro is Cbl responsive . Mutation of MCEE, the enzyme directly upstream of MUT in the propionate catabolic pathway, as well as in SUCLG1 or SUCLA2 the genes encoding the heterodimeric enzyme succinate‐CoA ligase immediately downstream of MUT, also result in disease including MMAuria of milder degree.…”

From structural biology to designing therapy for inborn errors of metabolism

“…This is illustrated in the example of methylmalonyl CoA mutase (MUT), one of the two destination enzymes requiring the vitamin B12 cofactor for catalysis, where to date >130 missense mutations are found on this 750-amino acid polypeptide to cause methylmalonic aciduria (MIM 251000). From a mapping of all known MUT mutations onto the protein crystal structure (Froese et al 2010), we selected 23 of them representative of diverse exonic regions, clinical phenotypes and ethnic populations, and performed a series of biochemical and cellular studies that characterize the protein stability (recombinant expression level, differential scanning fluorimetry) and enzyme catalysis (activity assay in patient cells) (Forny et al 2014) (Fig. 1).…”