Catherine L. Drennan

Cobalamin-dependent methionine synthase: the structure of a methylcobalamin-binding fragment and implications for other B12-dependent enzymes

Cobalamin-dependent methionine synthase is a large enzyme composed of structurally and functionally distinct regions. Recent studies have begun to define the roles of several regions of the protein. In particular, the structure of a 27 kDa cobalamin-binding fragment of the enzyme from Escherichia coli has been determined by X-ray crystallography, and has revealed the motifs and interactions responsible for recognition of the cofactor. The amino acid sequences of several adenosylcobalamin-dependent enzymes, the methylmalonyl coenzyme A mutases and glutamate mutases, show homology with the cobalamin-binding region of methionine synthase and retain conserved residues that are determinants for the binding of the prosthetic group, suggesting that these mutases and methionine synthase share common three-dimensional structures.

The reactivity of B12 cofactors: The proteins make a difference

Determination of the structure of intact methylmalonyl-CoA mutase from Propionibacterium shermanii, and comparisons with the structure of the cobalamin-binding fragment of methionine synthase from Escherichia coli, afford a first glimpse at the similarities and distinctions between the two principal classes of B12-dependent enzymes: the mutases and the methyltransferases.

A protein radical cage slows photolysis of methylcobalamin in methionine synthase from Escherichia coli

Methionine synthase from Escherichia coli is a B12-dependent enzyme that utilizes a methylcobalamin prosthetic group. In the catalytic cycle, the methyl group of methylcobalamin is transferred to homocysteine, generating methionine and cob(I)-alamin, and cob(I)alamin is then remethylated by a methyl group from methyltetrahydrofolate. Methionine synthase occasionally undergoes side reactions that produce the inactive cob(II)alamin form of the enzyme. One such reaction is photolytic homolysis of the methylcobalamin C-Co bond. Binding to the methionine synthase apoenzyme protects the methylcobalamin cofactor against photolysis, decreasing the rate of this reaction by approximately 50-fold. The X-ray structure of the cobalamin-binding region of methionine synthase suggests how the protein might protect the methylcobalamin cofactor in the resting enzyme. In particular, the upper face (methyl or beta face) of the cobalamin cofactor is in contact with several hydrophobic residues provided by an alpha-helical domain, and these residues could slow photolysis by caging the methyl radical and favoring recombination of the CH3./cob(II)alamin radical pair. We have introduced mutations at three positions in the cap domain; phenylalanine 708, phenylalanine 714, and leucine 715 have each been replaced by alanine. Calculations based on the wild-type structure predict that two of these three mutations (Phe708Ala and Leu715Ala) will increase solvent accessibility to the methylcobalamin cofactor, and in fact these mutations result in dramatic increases in the rate of photolysis. The third mutation, Phe714Ala, is not predicted to increase the accessibility of the cofactor and has only a modest effect on the photolysis rate of the enzyme. These results confirm that the alpha-helical domain covers the cofactor in the resting methylcobalamin enzyme and that residues from this domain can protect the enzyme against photolysis. Further, we show that binding the substrate methyltetrahydrofolate to the wild-type enzyme results in a saturable increase in the rate of photolysis, suggesting that substrate binding induces a conformational change in the protein that increases the accessibility of the methylcobalamin cofactor.