Evolution of enzymatic activity in the enolase superfamily: Structure of o-succinylbenzoate synthase from Escherichia coli in complex with Mg²⁺ and o-succinylbenzoate

The X-ray structures of the ligand free (apo) and the Mg²⁺·o-succinylbenzoate (OSB) product complex of o-succinylbenzoate synthase (OSBS) from Escherichia coli have been solved to 1.65 and 1.77 Å resolution, respectively. The structure of apo OSBS was solved by multiple isomorphous replacement in space group P2₁2₁2₁; the structure of the complex with Mg²⁺·OSB was solved by molecular replacement in space group P2₁2₁2. The two domain fold found for OSBS is similar to those found for other members of the enolase superfamily: a mixed α/β capping domain formed from segments at the N- and C-termini of the polypeptide and a larger (β/α)₇β barrel domain. Two regions of disorder were found in the structure of apo OSBS: (i) the loop between the first two β-strands in the α/β domain; and (ii) the first sheet - helix pair in the barrel domain. These regions are ordered in the product complex with Mg²⁺·OSB. As expected, the Mg²⁺·OSB pair is bound at the C-terminal end of the barrel domain. The electron density for the phenyl succinate component of the product is well-defined; however, the 1-carboxylate appears to adopt multiple conformations. The metal is octahedrally coordinated by Asp¹⁶¹, Glu¹⁹⁰, and Asp²¹³, two water molecules, and one oxygen of the benzoate carboxylate group of OSB. The loop between the first two β-strands in the α/β motif interacts with the aromatic ring of OSB. Lys¹³³ and Lys²³⁵ are positioned to function as acid/base catalysts in the dehydration reaction. Few hydrogen bonding or electrostatic interactions are involved in the binding of OSB to the active site; instead, most of the interactions between OSB and the protein are either indirect via water molecules or via hydrophobic interactions. As a result, evolution of both the shape and the volume of the active site should be subject to few structural constraints. This would provide a structural strategy for the evolution of new catalytic activities in homologues of OSBS and a likely explanation for how the OSBS from Amycolaptosis also can catalyze the racemization of N-acylamino acids [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258].