A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

Abstract

B12-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-Lmethionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide crosslinking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reduces the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme. multimodular protein ͉ protein conformation ͉ vitamin B12

Insights into the reactivation of cobalamin-dependent methionine synthase

“…However, crystallographic refinement consistently resulted in a long distance (Ϸ3.4 Å) from the cobalt to N2 of H759. The most likely explanation for the long bond is photoreduction of the Co(III) in the X-ray beam, as seen in other Co(III)Cbl structures (12,21). Density for the Y1139 side chain indicates partial disorder, but nonetheless clear evidence of movement away from the cobalamin toward the empty AdoHcy/AdoMet binding pocket.…”

“…However, density near the upper axial position was assigned to a water molecule stabilized by a hydrogen bond with the side chain of E1097. Compared to the previously determined structure of the MeCo(III)Cbl form (12), the side chain of E1097 is repositioned by AdoHcy binding, allowing formation of a hydrogen bond to the water molecule. Moreover, the distance between the cobalt and the Y1139 OH moiety has decreased from 4.5 Å to 4.0 Å.…”

“…To reduce conformational flexibility a disulfide cross-link was introduced by mutating Ile-690 and Gly-743 to cysteines ( S-S MetH CT ). The X-ray structure of the MeCo(III)Cbl form of S-S MetH CT revealed that the MeCo(III)Cbl cofactor had been photolyzed by the X-ray beam during data collection, and that the cobalt appeared 4-coordinate with no electron density evident for the methyl group (12). Four-coordinate Co(II)Cbl has never been observed in aqueous solution.…”

“…Although the structure of the full-length enzyme has not yet been determined, there are structures of the individual cobalamin-binding (9) and AdoMet-binding modules (10) from E. coli. In addition, 2 multimodular fragments have been characterized: an N-terminal polypeptide (Hcy and CH 3 -H 4 folate modules) from Thermotoga maritima (11,12) and a C-terminal polypeptide (cobalamin and AdoMet modules) from E. coli (12,13). During catalysis the enzyme cycles between conformations in which the cobalamin-binding module is juxtaposed alternatively with the Hcy-binding module and the CH 3 -H 4 folatebinding module, whereas for the reductive reactivation the enzyme assumes a conformation in which the cobalamin-binding module is juxtaposed with the AdoMet-binding module.…”

Two distinct pools of B12analogs reveal community interdependencies in the ocean

“…To compare cobalamin-and pseudocobalamin-binding sites in MetH, we gathered MetH amino acid sequences from organisms known to use true cobalamin or pseudocobalamin as their cofactor. We then aligned the sequences and used a known crystal structure (32,53) to visualize the binding pocket.…”