MMUT Clinical Variants Define a Goldilocks Zone for B12-Based Radical Chemistry

Abstract

MMUT is one of only two enzymes in the human proteome that depend on the B12 cofactor for activity. It catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA using 5′-deoxyadenosylcobalamin (AdoCbl) as a radical generator. Clinical mutations in MMUT are inherited in an autosomal recessive manner and are classified as B12-responsive mut– and B12 unresponsive mut0 subgroups. In this study, we report that missense mutations located at the same residue, i.e., Ala-137, are associated with distinct biochemical penalties. While the A137G mutation disables cofactor loading from the MMAB chaperone, it shows an ∼700-fold weaker affinity for binding AdoCbl from solution and exhibits an ∼700-fold lower activity than the wild-type enzyme. In contrast, the A137V variant binds AdoCbl tightly, can be partially loaded with cofactor from MMAB from solution, but exhibits ∼5000-fold lower activity, consistent with its mut0 classification. Despite the overall similarity in the crystal structure with wild-type MMUT, a close-up of the A137V active site reveals profound differences in the 5′-deoxyadenosyl moiety, which is held in a previously unseen pose. Structural analyses predict that while Gly-137 is too small to correctly position Tyr-110, critical for transition state stabilization, Val-137 is too large to even accommodate it, exemplifying the catalysis-permissive Goldilocks zone at this site. Our study predicts that the A137G variant might belong to the mut– subgroup and exhibit therapeutic responsiveness to a high dose of B12.