o-Succinylbenzoate synthase (OSBS) from Escherichia coli, a member of the enolase superfamily, catalyzes an exergonic dehydration reaction in the menaquinone biosynthetic pathway in which 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) is converted to 4-(2′-carboxyphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB). Our previous structural studies of the Mg2+·OSB complex established that OSBS is a member of the muconate lactonizing enzyme subgroup of the superfamily: the essential Mg2+ is coordinated to carboxylate ligands at the ends of the third, fourth, and fifth β-strands of the (β/α)7β-barrel catalytic domain, and the OSB product is located between the Lys 133 at the end of the second β-strand and the Lys 235 at the end of the sixth β-strand [Thompson, T. B., Garrett, J. B., Taylor, E. A, Meganathan, R., Gerlt, J. A., and Rayment, I. (2000) Biochemistry 39, 10662-76]. Both Lys 133 and Lys 235 were separately replaced with Ala, Ser, and Arg residues; all six mutants displayed no detectable catalytic activity. The structure of the Mg2+·SHCHC complex of the K133R mutant has been solved at 1.62 Å resolution by molecular replacement starting from the structure of the Mg2+·OSB complex. This establishes the absolute configuration of SHCHC: the C1-carboxylate and the C6-OH leaving group are in a trans orientation, requiring that the dehydration proceed via a syn stereochemical course. The side chain of Arg 133 is pointed out of the active site so that it cannot function as a general base, whereas in the wild-type enzyme complexed with Mg2+·OSB, the side chain of Lys 133 is appropriately positioned to function as the only acid/base catalyst in the syn dehydration. The ε-ammonium group of Lys 235 forms a cation-π interaction with the cyclohexadienyl moiety of SHCHC, suggesting that Lys 235 also stabilizes the enediolate anion intermediate in the syn dehydration via a similar interaction.
ASJC Scopus subject areas