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-succinylbenzo nte (OSB) product complex of o-succinylbenzoate synthase (OSBS) from Escher ichia coli have been solved to 1.65 and 1.77 Angstrom resolution, respectiv ely. The structure of apo OSBS was solved by multiple isomorphous replaceme nt in space group P2(1)2(1)2(1); the structure of the complex with Mg2+. OS B was solved by molecular replacement in space group P2(1)2(1)2. The two do main fold found for OSBS is similar to those found for other members of the enolase superfamily: a mixed alpha/beta capping domain formed from segment s at the N- and C-termini of the polypeptide and a larger (alpha/beta)(7)be ta barrel domain. Two regions of disorder were found in the structure of ap o OSBS: (i) the loop between the first two beta-strands in the alpha/beta d omain; and (ii) the first sheet-helix pair in the barrel domain. These regi ons 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 electr on density for the phenyl succinate component of the product is well-define d; however, the 1-carboxylate appears to adopt multiple conformations. The metal is octahedrally coordinated by Asp(161), Glu(190), and Asp(213), two water molecules, and one oxygen of the benzoate carboxylate group of OSB. T he loop between the first two beta-strands in the alpha/beta motif interact s with the aromatic ring of OSB, Lys(133) and Lys(235) are positioned to fu nction as acid/base catalysts in the dehydration reaction. Few hydrogen bon ding or electrostatic interactions are involved in the binding of OSB to th e active site; instead, most of the interactions between OSB and the protei n an either indirect via water molecules or via hydrophobic interactions. A s a result, evolution of both the shape and the volume of the active site s hould be subject to few structural constraints. This would provide a struct ural strategy for the evolution of new catalytic activities in homologues o f OSBS and a likely explanation for how the OSBS from Amycolaptosis also ca n 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) B iochemistry 38, 4252-4258].