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

Abstract

Mutations in the human MMAA gene cause the metabolic disorder cblA-type methylmalonic aciduria (MMA), although knowledge of the mechanism of dysfunction remains lacking. MMAA regulates the incorporation of the cofactor adenosylcobalamin (AdoCbl), generated from the MMAB adenosyltransferase, into the destination enzyme methylmalonyl-CoA mutase (MUT). This function of MMAA depends on its GTPase activity, which is stimulated by an interaction with MUT. Here, we present 67 new patients with cblA-type MMA, identifying 19 novel mutations. We biochemically investigated how missense mutations in MMAA in 22 patients lead to disease. About a third confer instability to the recombinant protein in bacterial and human expression systems. All 15 purified mutant proteins demonstrated wild-type like intrinsic GTPase activity and only one (p.Asp292Val), where the mutation is in the GTP binding domain, revealed decreased GTP binding. However, all mutations strongly decreased functional association with MUT by reducing GTPase activity stimulation upon incubation with MUT, while nine mutant proteins additionally lost the ability to physically bind MUT. Finally, all mutations interfered with gating the transfer of AdoCbl from MMAB to MUT. This work suggests loss of functional interaction between MMAA and MUT as a disease-causing mechanism that impacts processing and assembly of a cofactor to its destination enzyme.

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

“…45 This transfer, however, is gated by a third protein methylmalonic aciduria cblA type (MMAA), which additionally appears to protect the AdoCbl from oxidation during the MUT catalytic cycle in a guanosine triphosphate GTP dependent manner. 46,47 Mutation of MMAA in patients is described as the cblA defect, 43 and every patient mutation described to date either results in a complete loss of MMAA protein or interferes with the interaction between MMAA and MUT. 46 MUT itself catalyzes the rearrangement of L-methylmalonyl-CoA to succinyl-CoA in an AdoCbl dependent manner.…”

“…46,47 Mutation of MMAA in patients is described as the cblA defect, 43 and every patient mutation described to date either results in a complete loss of MMAA protein or interferes with the interaction between MMAA and MUT. 46 MUT itself catalyzes the rearrangement of L-methylmalonyl-CoA to succinyl-CoA in an AdoCbl dependent manner. This pathway is an extension of the propionate catabolic pathway, which is required for the breakdown of the branched-chain amino acids valine, isoleucine, methionine and threonine, odd-chain fatty acids and the side chain of cholesterol, and whose product enters the tricarboxylic acid cycle as an anapleurotic substrate.…”

FoldX as Protein Engineering Tool: Better Than Random Based Approaches?

“…FoldX calculation were performed 5 times and averaged for each mutation. 8 6 Highest destabilization (−ΔT m = −8.8 °C) 1.55 Å [ 114 ] Protein stabilization Growth factor 2 ( Homo sapiens ) Cut off (ΔΔG < −1 kcal mol − 1) 5 2 Stabilization ΔT m = 3.7 °C 1.6 Å [ 115 ] Flavin mononucleotide based fluorescent Protein ( Bacillus subtilis ) Cut off (ΔΔG < −1 kcal mol −1 ) performed additionally further pre-selections 22 15 Stabilization ΔT m = 11.4 °C Resolution under 2.2 Å [ 116 ] Endolysin PlyC ( Bacteriophage ) Cut off (ΔΔG < −1 kcal mol −1 ) 92 mutants were determined by FoldX and reduced by visual inspection and by Rosetta 12 3 Stabilization ΔT m = 2.2 °C 3.3 Å refined using Rosetta Relax [ 117 ] Database (known mutations) Cut off value ΔΔG < −1.0 kcal mol −1 30 20 n.d. 1.5 to 2.25 Å [ 87 ] Protein destabilization Repair protein MSH2 ( Homo sapiens ) Cut off value (ΔΔG > 5 kcal mol −1 ) 24 22 Destabilization of >3 kcal mol −1 3.3 Å [ 85 ] cblA -Type methylmalonic aciduria ( Homo sapiens ) Cut off value ΔΔG between 3.48 and 11.15 kcal mol −1 22 22 – 2.64 Å [ 118 ] Investigation of proline influence on stability Fungal chimeric cellobiohydrolase Cel6A ( Humicola insolens ) ΔΔG of exchanges of wild type amino acid against Pro exchange was calculated ...…”

Methylmalonic acidemia: Current status and research priorities

“…MCM, an adenosylcobalamin-dependent enzyme, have a function in isomerization of L-methylmalonyl-CoA to succinyl-CoA, for entry into the tricarboxylic acid cycle for energy production. All mutations of MMAA strongly decreased functional association with MUT and interfered with gating the transfer of AdoCbl from MMAB to MUT, which is a disease-causing mechanism of cblA type MMA (6). The MMAB gene encodes the mitochondrial enzyme ATP: cobalamin adenosyl transferase (ATR), which catalyzes transfer of an adenosyl group from ATP to cobalamin (I) to form AdoCbl.…”