HOW A PROTEIN PREPARES FOR B-12 BINDING - STRUCTURE AND DYNAMICS OF THE B-12-BINDING SUBUNIT OF GLUTAMATE MUTASE FROM CLOSTRIDIUM-TETANOMORPHUM

Background: Glutamate mutase is an adenosylcobamide (coenzyme B-12) de pendent enzyme that catalyzes the reversible rearrangement of (2S)-glu tamate to (2S,3S)-3-methylaspartate. The enzyme from Clostridium tetan omorphum comprises two subunits (of 53.7 and 14.8 kDa) and in its acti ve form appears to be an alpha(2)beta(2) tetramer. The smaller subunit , termed MutS, has been characterized as the B-12-binding component. K nowledge an the structure of a B-12-binding apoenzyme does not exist. Results: The solution structure and important dynamical aspects of Mut S have been determined from a heteronuclear NMR study. The global fold of MutS in solution resembles that determined by X-ray crystallograph y for the B-12-binding domains of Escherichia coli methionine synthase and Propionibacterium shermanii methylmalonyl CoA mutase. In these tw o proteins a histidine residue displaces the endogenous cobalt-coordin ating ligand of the B-12 cofactor. In MutS, however, the segment of th e protein containing the conserved histidine residue forms part of an unstructured and mobile extended loop. Conclusions: A comparison of th e crystal structures of two B-12-binding domains, with bound B-12 cofa ctor, and the solution structure of the apoprotein MutS has helped to clarify the mechanism of B-12 binding. The major part of MutS is preor ganized for B-12 finding, but the B-12-binding site itself is only par tially formed. Upon binding B-12, important elements of the binding si te appear to become structured, including an a helix that forms one si de of the cleft accommodating the nucleotide 'tail' of the cofactor.