Structure of MMACHC Reveals an Arginine-Rich Pocket and a Domain-Swapped Dimer for Its B₁₂ Processing Function

Abstract

Defects in the MMACHC gene represent the most common disorder of cobalamin (Cbl) metabolism, affecting synthesis of the enzyme cofactors adenosyl-Cbl and methyl-Cbl. The encoded MMACHC protein binds intracellular Cbl derivatives with different upper axial ligands and exhibits flavin mononucleotide (FMN)-dependent decyanase activity toward cyano-Cbl as well as glutathione (GSH)-dependent dealkylase activity toward alkyl-Cbls. We determined the structure of human MMACHC·adenosyl-Cbl complex, revealing a tailor-made nitroreductase scaffold which binds adenosyl-Cbl in a "base-off, five-coordinate" configuration for catalysis. We further identified an arginine-rich pocket close to the Cbl binding site responsible for GSH binding and dealkylation activity. Mutation of these highly conserved arginines, including a replication of the prevalent MMACHC missense mutation, Arg161Gln, disrupts GSH binding and dealkylation. We further showed that two Cbl-binding monomers dimerize to mediate the reciprocal exchange of a conserved "PNRRP" loop from both subunits, serving as a protein cap for the upper axial ligand in trans and required for proper dealkylation activity. Our dimeric structure is supported by solution studies, where dimerization is triggered upon binding its substrate adenosyl-Cbl or cofactor FMN. Together our data provide a structural framework to understanding catalytic function and disease mechanism for this multifunctional enzyme.

Structure of Human B12 Trafficking Protein CblD Reveals Molecular Mimicry and Identifies a New Subfamily of Nitro-FMN Reductases

“…The flavin-and substrate-binding sites are located at the dimer interface in these proteins. In contrast, CblD and CblC do not form dimers in solution, and the dimerization interface seen in their crystal structures is distinct from that of other NFR family members in that it does not involve the long helix (28,42).…”

“…6, A and B). Solution of the crystal structure of CblC led to the recognition that it resembles some members in the NFR superfamily (28,42). CblC and CblD share an identical core of antiparallel ␤-sheets with similar structural elements in comparable arrangements framing the core.…”

Proteomics of vitamin B12 processing

“…Studies with the bovine isoform of MMACHC revealed that the reduced form of glutathione stabilizes MMACHC, suggesting that intracellular redox control could play a role in the regulation of the protein ' s lifetime [ 41 -44 ]. Koutmos [ 3 ] and Froese [ 45 ] glutathione-binding pocket up above the β -axial ligand position [ 45 ]. Importantly, the arginine-rich pocket comprises residues Arg161, Arg206 and Arg230 ( recombinant mutant Arg206Gln was insoluble suggesting a structural role for this residue, and that mutants Arg161Gln and Arg230Gln abolished GSH binding and dealkylase activity, which is a strong indication that these amino acid residues are critical for GSH binding [ 45 ].…”

“…Koutmos [ 3 ] and Froese [ 45 ] glutathione-binding pocket up above the β -axial ligand position [ 45 ]. Importantly, the arginine-rich pocket comprises residues Arg161, Arg206 and Arg230 ( recombinant mutant Arg206Gln was insoluble suggesting a structural role for this residue, and that mutants Arg161Gln and Arg230Gln abolished GSH binding and dealkylase activity, which is a strong indication that these amino acid residues are critical for GSH binding [ 45 ]. The authors noted that FMN, and to a lesser extent Cbl, induces the dimerization of MMACHC, a previously unrecognized feature of the protein [ 45 ].…”

Novel Deletion Mutation Identified in a Patient with Late-Onset Combined Methylmalonic Acidemia and Homocystinuria, cblC Type

“…Furthermore, MMACHC has been proposed to interact with combined methylmalonic aciduria and homocystinuria cblD type (MMADHC), the downstream protein in the cobalamin pathway (Deme et al 2012;Plesa et al 2011). Recently the three-dimensional structure of human MMACHC was determined (Froese et al 2012;Koutmos et al 2011). This provides a structural framework to understand the effects of MMACHC mutations and gives new biochemical insight into the catalytic functions of MMACHC.…”