The B12-Binding Subunit of Glutamate Mutase from Clostridium tetanomorphum Traps the Nucleotide Moiety of Coenzyme B12

Glutamate mutase from Clostridium tetanomorphum binds coenzyme B12 in a base-off/His-on form, in which the nitrogenous ligand of the B12-nucleotide function is displaced from cobalt by a conserved histidine. The effect of binding the B12-nucleotide moiety to MutS, the B12-binding subunit of glutamate mutase, was investigated using NMR spectroscopic methods. Binding of the B12-nucleotide to MutS was determined to occur with Kd=5.6(±0.7) mM and to be accompanied by a specific conformational change in the protein. The nucleotide binding cleft of the apo-protein, which is formed by a dynamic segment with propensity for partial α-helical conformation (the “nascent” α-helix), becomes completely structured upon binding of the B12-nucleotide, with formation of helix α1. In contrast, the segment containing the conserved residues of the B12-binding Asp-x-His-x-x-Gly motif remains highly dynamic in the protein/B12-nucleotide complex. From relaxation studies, the time constant τ, which characterizes the time scale for the formation of helix α1, was estimated to be about 30 μs 15N and was the same in both, apo-protein and nucleotide-bound protein. Thus, the binding of the B12-nucleotide moiety does not significantly alter the kinetics of helix formation, but only shifts the equilibrium towards the structured fold. These results indicate MutS to be structured in such a way, as to be able to trap the nucleotide segment of the base-off form of coenzyme B12 and provide, accordingly, the first structural clues as to how the process of B12-binding occurs.