HOW COENZYME B₁₂ RADICALS ARE GENERATED: METHYLMALONYL-COA MUTASE AT 2Å RESOLUTION. P.R. Evans and F. Mancia - MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

This structure shows how the enzyme catalyses the formation of the adenosyl radical from coenzyme B₁₂. Methylmalonyl-CoA mutase is a member of a class of enzymes that bind the cobalt-containing 5'-deoxyadenosyl-cobalamin cofactor (coenzyme B₁₂) and catalyse 1,2 intramolecular rearrangements in which a hydrogen atom is exchanged with a group on an adjacent carbon. Methylmalonyl-CoA mutase catalyses the interconversion between (2R)-methylmalonyl-CoA and succinyl-CoA. Such reactions involve radical intermediates: the initial radical arises from the homolysis of the unique Co-C bond of coenzyme B₁₂, amongst the very few metal-carbon bond known in nature. A long-standing puzzle has been how the protein weakens this Co-C bond towards homolytic cleavage.

Methylmalonyl-CoA mutase is the only adenosylcobalamin-dependent enzyme present in both animals and microrganisms. In the bacterium Propionibacterium shermanii it is a key enzyme in the fermentation to propionate, whilst in mammalian liver it is responsible for the conversion of odd-chain fatty acids and branched-chain amino acids to succinyl-CoA for further degradation. The P.shermanii enzyme is an [[alpha]],[[beta]] heterodimer of 150kD total molecular weight with one active site per dimer. We have solved the structure of the ternary complex between the recombinant protein expressed in E.coli, coenzyme B₁₂, and the partial substrate desulpho-CoA (coenzyme A with the final sulphur atom replaced by a hydrogen).

Each subunit has essentially a two domain architecture. In the catalytic [[alpha]] chain, the B₁₂ is sandwiched between a C-terminal flavodoxin [[alpha]]/[[beta]] domain and an N-terminal [[beta]]/[[alpha]] TIM barrel. A conserved histidine from the flavodoxin-like domain provides axial coordination to the cobalt atom in a very similar way to that seen in methionine synthase. The histidine-cobalt distance is very long (2.5Å compared to 1.95-2.2Å in free cobalamins), suggesting that the enzyme positions the histidine in order to weaken the Co-C of the cofactor and favour the formation of the initial radical The substrate is bound through a hole along the axis of the barrel, pointing into a deeply buried active site on the 5'-deoxyadenosyl or catalytic side of the B₁₂.