Structure and dynamics of the B 12 ‐binding subunit of glutamate mutase from Clostridium cochlearium

Glutamate mutase (Glm) is an adenosylcobamide‐dependent enzyme that catalyzes the reversible rearrangement of (2 S )‐glutamate to (2 S ,3 S )‐3‐methylaspartate. The active enzyme from Clostridium cochlearium consists of two subunits (of 53.6 and 14.8 kDa) as an α 2 β 2 tetramer, whose assembly is mediated by coenzyme B 12 . The smaller of the protein components, GlmS, has been suggested to be the B 12 ‐binding subunit. Here we report the solution structure of GlmS, determined from a heteronuclear NMR‐study, and the analysis of important dynamical aspects of this apoenzyme subunit. The global fold and dynamic behavior of GlmS in solution are similar to those of the corresponding subunit MutS from C. tetanomorphum , which has previously been investigated using NMR‐spectroscopy. Both solution structures of the two Glm B 12 ‐binding subunits share striking similarities with that determined by crystallography for the B 12 ‐binding domain of methylmalonyl CoA mutase (Mcm) from Propionibacterium shermanii , which is B 12 bound. In the crystal structure a conserved histidine residue was found to be coordinated to cobalt, displacing the endogenous axial ligand of the cobamide. However, in GlmS and MutS the sequence motif, Asp‐x‐His‐x‐x‐Gly, which includes the cobalt‐coordinating histidine residue, and a predicted α‐helical region following the motif, are present as an unstructured and highly mobile loop. In the absence of coenzyme, the B 12 ‐binding site apparently is only partially formed. By comparing the crystal structure of Mcm with the solution structures of B 12 ‐free GlmS and MutS, a consistent picture on the mechanism of B 12 binding has been obtained. Important elements of the binding site only become structured upon binding B 12 ; these include the cobalt‐coordinating histidine residue, and an α helix that forms one side of the cleft accommodating the nucleotide ‘tail’ of the coenzyme.