How a Protein Binds B ₁₂ : A 3.0 Å X-Ray Structure of B ₁₂ -Binding Domains of Methionine Synthase

Abstract

The crystal structure of a 27-kilodalton methylcobalamin-containing fragment of methionine synthase from Escherichia coli was determined at 3.0 A resolution. This structure depicts cobalamin-protein interactions and reveals that the corrin macrocycle lies between a helical amino-terminal domain and an alpha/beta carboxyl-terminal domain that is a variant of the Rossmann fold. Methylcobalamin undergoes a conformational change on binding the protein; the dimethylbenzimidazole group, which is coordinated to the cobalt in the free cofactor, moves away from the corrin and is replaced by a histidine contributed by the protein. The sequence Asp-X-His-X-X-Gly, which contains this histidine ligand, is conserved in the adenosylcobalamin-dependent enzymes methylmalonyl-coenzyme A mutase and glutamate mutase, suggesting that displacement of the dimethylbenzimidazole will be a feature common to many cobalamin-binding proteins. Thus the cobalt ligand, His759, and the neighboring residues Asp757 and Ser810, may form a catalytic quartet, Co-His-Asp-Ser, that modulates the reactivity of the B12 prosthetic group in methionine synthase.

Native Corrinoids fromClostridium cochleariumAre Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B12and Factor A

“…This situation is drastically different from that in the benzimidazolylcobamides, where H bonding and protonation of the nucleotide base cannot directly occur in the cobalt-coordinated state. A mechanism for the control of the organometallic reactivity of protein-bound corrinoid cofactors involves weakening of the cobalt coordination of the axial transligand, conveyed (in part) by H bonding it to other protein residues in "regulatory" diads or triads (21,43,51,52). Only in their base-off-His-on form can bound benzimidazolylcobamides be set to participate directly in such H-bonded regulatory units via a coordinated histidine.…”

“…On the other hand, the bound corrinoid in the corrinoidiron-sulfur protein involved in the synthesis of acetyl-coenzyme A from Clostridium thermoaceticum was indicated by spectroscopic means to be "base-off," i.e., to have the nucleotide base of the corrinoid decoordinated in the protein (57). A pioneering structure analysis by Drennan et al of the B 12 -binding domain of a methionine synthase from E. coli that depends upon methylcobalamin as a cofactor (21,51) uncovered the "base-off-His-on" form of the protein-bound complete methylcorrinoid, in which a proteinic histidine residue displaces the cobalt-coordinating DMB base of the corrinoid cofactor. The relevance of the base-off-His-on mode of binding of corrinoids in cobamide-dependent enzymes (46,72) was supported further by two additional crystal structures, of methylmalonyl coenzyme A mutase (52) and glutamate mutase (58), which both depend upon an adenosylcobamide as a cofactor, such as coenzyme B 12 (5Ј-deoxy-5Ј-adenosylcobalamin).…”

Homology modeling of human methylmalonyl‐CoA mutase: A structural basis for point mutations causing methylmalonic aciduria

“…In particular, a cluster of such mutations was noted near the C-terminus of the protein (Crane & Ledley,I994), which is now recognized as the common cobalamin-binding domain of many B ,,-binding enzymes (Marsh & Holloway, 1992;Drennan et al, 1994;Mancia et al, 1996). Intriguingly studies with somatic cell hybrids have demonstrated that a number of these mutations show interallelic complementation with a mutation (R93H) in the N-terminal region of MCM.…”

“…3). The structure of the cobalamin-binding domain was first recognized for methionine synthase (Drennan et al, 1994) …”

“…shermanii MCM) is another conserved residue essential to create the dimethylbenzimidazole-binding pocket (Drennan et al, 1994 2"hydroxy group of the ribose ring in the pseudo-nucleotide tail, and the mutation G703R (mut") would clearly lead to severe steric clashes and the introduction of a positive charge into a buried environment.…”