Abstract
Pathogenic variants in MMAB cause cblB-type methylmalonic aciduria, an autosomal-recessive disorder of propionate metabolism. MMAB encodes ATP:cobalamin adenosyltransferase, using ATP and cob(I)alamin to create 5’-deoxyadenosylcobalamin (AdoCbl), the cofactor of methylmalonyl-CoA mutase (MMUT). We identified bi-allelic disease-causing variants in MMAB in 97 individuals with cblB-type methylmalonic aciduria, including 33 different and 16 novel variants. Missense changes accounted for the most frequent pathogenic alleles (p.(Arg186Trp), N = 57; p.(Arg191Trp), N = 19); while c.700C > T (p.(Arg234*)) was the most frequently identified truncating variant (N = 14). In fibroblasts from 76 affected individuals, the ratio of propionate incorporation in the presence and absence of hydroxocobalamin (PI ratio) was associated to clinical cobalamin responsiveness and later disease onset. We found p.(Arg234*) to be associated with cobalamin responsiveness in vitro, and clinically with later onset; p.(Arg186Trp) and p.(Arg191Trp) showed no clear cobalamin responsiveness and early onset. Mapping these and novel variants onto the MMAB structure revealed their potential to affect ATP and AdoCbl binding. Follow-up biochemical characterization of recombinant MMAB identified its three active sites to be equivalent for ATP binding, determined by fluorescence spectroscopy (Kd = 21 µM) and isothermal calorimetry (Kd = 14 µM), but function as two non-equivalent AdoCbl binding sites (Kd1 = 0.55 μM; Kd2 = 8.4 μM). Ejection of AdoCbl was activated by ATP (Ka = 24 µM), which was sensitized by the presence of MMUT (Ka = 13 µM). This study expands the landscape of pathogenic MMAB variants, provides association of in vitro and clinical responsiveness, and facilitates insight into MMAB function, enabling better disease understanding.
Highlights
The human ATP:cob(I)alamin adenosyltransferase enzyme (MMAB; EC 2.5.1.17) is a ubiquitously expressed protein involved in intracellular cobalamin metabolism
Its role involves the adenosylation of cob(I)alamin by ATP, creating adenosylcobalamin (AdoCbl), the cofactor of methylmalonyl-CoA mutase (MMUT; EC 5.4.99.2)
We further show data indicating that biochemical parameters of MMAB, including ATP and AdoCbl affinity, differ from those of its structural homologs, which may influence the functional interaction of MMAB with downstream effectors such as MMUT and MMAA
Summary
The human ATP:cob(I)alamin adenosyltransferase enzyme (MMAB; EC 2.5.1.17) is a ubiquitously expressed protein involved in intracellular cobalamin (vitamin B12) metabolism. Its role involves the adenosylation of cob(I)alamin by ATP, creating adenosylcobalamin (AdoCbl), the cofactor of methylmalonyl-CoA mutase (MMUT; EC 5.4.99.2) This latter enzyme catalyzes the anaplerotic conversion of methylmalonyl-CoA to succinyl-CoA as part of the catabolism. Bi-allelic pathogenic variants in the MMAB gene (OMIM #607,568) cause isolated methylmalonic aciduria of the cblB-type (MMA; OMIM #251,110). This disease can present with an early-onset severe form (presentation during the first month of life), whose symptoms include severe metabolic ketoacidosis, caused by accumulating acids (including methylmalonic acid) as well as lethargy potentially deteriorating to coma, which is partly owed to raised plasma ammonia levels (Forny et al 2021). Confirmation of diagnosis of cblB-type MMA classically involves assessment of residual propionate pathway function in patient fibroblasts by measuring propionate incorporation activity (PI activity) – a diagnostic assay which uses radio-labelled
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