Functional characterization and categorization of missense mutations that cause methylmalonyl-CoA 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.

Keywords: MUT; cobalamin; methylmalonic aciduria; methylmalonyl-CoA mutase; thermolability.

Figure 1

Structural mapping of MUT missense mutations. A: The human MUT structure in complex with MCoA and AdoCbl (PDB code 2XIQ) is colored according to domains, that is, N‐terminal substrate‐binding domain cyan, C‐terminal cofactor‐binding domain magenta, and interdomain linker yellow. Mutations in this study are shown as red circles (mut ⁰) or blue circles (mut ⁻). Ligands are in stick representation, colored white for MCoA and black for AdoCbl. B: Domain organization of MUT highlighting locations of the studied mutations, dimerization interface, and binding regions for MCoA and AdoCbl in the polypeptide. An interactive version of this structural representation is available online at www.thesgc.org/MUT.

Figure 2

MUT missense mutations confer different effects on stability and activity. A: Coomassie staining of SDS‐PAGE following small‐scale bacterial expression and affinity purification. For each mutation, lanes for total cell lysate (“L,” left) and eluant after purification (“E,” right) are shown. B: Western blot following expression of each mutation in a MUT‐deficient patient cell line. Vector, empty vector. C: Total MUT activity (assay with 50 μM AdoCbl) of each mutation expressed as percent of mean wt activity. D: K _M for AdoCbl, expressed as times the mean wt value. E: ∆T _m of apo mutant compared to apo‐wt protein (see Fig. 3). –, not applicable.

Figure 3

Thermolability of MUT mutations. The change in T _m values (ΔT _m, compared with apo wt) for each mutant MUT in the apo state, or following the addition of AdoCbl alone or with AdoCbl and MCoA, is shown. Black, apo protein; red, with 50 μM AdoCbl; blue, with 50 μM AdoCbl and 500 μM MCoA. Error bars depict SEM from at least three measurements. (Inset) Representative DSF melting curves of wt MUT in the apo form (black), with 50 μM AdoCbl (red) and with 50 μM AdoCbl and 500 μM MCoA (blue).

Figure 4

Stabilization of mutant MUT by osmolytes. Representative DSF melting curves for p.P86L (A) and p.G426R (B) mutants in the apo form (gray) and in the presence of osmolytes (colors) are shown along with wt MUT (black).