Versatile enzymology and heterogeneous phenotypes in cobalamin complementation type C disease.

Nutritional deficiency and genetic errors that impair the transport, absorption, and utilization of vitamin B12 (B12) lead to hematological and neurological manifestations. The cblC disease (cobalamin complementation type C) is an autosomal recessive disorder caused by mutations and epi-mutations in the MMACHC gene and the most common inborn error of B12 metabolism. Pathogenic mutations in MMACHC disrupt enzymatic processing of B12, an indispensable step before micronutrient utilization by the two B12-dependent enzymes methionine synthase (MS) and methylmalonyl-CoA mutase (MUT). As a result, patients with cblC disease exhibit plasma elevation of homocysteine (Hcy, substrate of MS) and methylmalonic acid (MMA, degradation product of methylmalonyl-CoA, substrate of MUT). The cblC disorder manifests early in childhood or in late adulthood with heterogeneous multi-organ involvement. This review covers current knowledge on the cblC disease, structure-function relationships of the MMACHC protein, the genotypic and phenotypic spectra in humans, experimental disease models, and promising therapies.

Thiolatocobalamins repair the activity of pathogenic variants of the human cobalamin processing enzyme CblC.

Thiolatocobalamins are a class of cobalamins comprised of naturally occurring and synthetic ligands. Glutathionylcobalamin (GSCbl) occurs naturally in mammalian cells, and also as an intermediate in the glutathione-dependent dealkylation of methylcobalamin (MeCbl) to form cob(I)alamin by pure recombinant CblC from C. elegans. Glutathione-driven deglutathionylation of GSCbl was demonstrated both in mammalian as well as in C. elegans CblC. Dethiolation is orders of magnitude faster than dealkylation of Co-C bonded cobalamins, which motivated us to investigate two synthetic thiolatocobalamins as substrates to repair the enzymatic activity of pathogenic CblC variants in humans. We report the synthesis and kinetic characterization of cysteaminylcobalamin (CyaCbl) and 2-mercaptopropionylglycinocobalamin (MpgCbl). Both CyaCbl and MpgCbl were obtained in high purity (90-95%) and yield (78-85%). UV-visible spectral properties agreed with those reported for other thiolatocobalamins with absorbance maxima observed at 372 nm and 532 nm. Both CyaCbl and MpgCbl bound to wild type human recombinant CblC inducing spectral blue-shifts characteristic of the respective base-on to base-off transitions. Addition of excess glutathione (GSH) resulted in rapid elimination of the ß-ligand to give aquacobalamin (H2OCbl) as the reaction product under aerobic conditions. Further, CyaCbl and MpgCbl underwent spontaneous dethiolation thereby repairing the loss of activity of pathogenic variants of human CblC, namely R161G and R161Q. We posit that thiolatocobalamins could be exploited therapeutically for the treatment of inborn errors of metabolism that impair processing of dietary and supplemental cobalamin forms. While these disorders are targets for newborn screening in some countries, there is currently no effective treatment available to patients.