Evolution of enzymatic activity in the enolase superfamily: structural and mutagenic studies of the mechanism of the reaction catalyzed by o-succinylbenzoate synthase from Escherichia coli.

Abstract

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 Mg(2+).OSB complex established that OSBS is a member of the muconate lactonizing enzyme subgroup of the superfamily: the essential Mg(2+) is coordinated to carboxylate ligands at the ends of the third, fourth, and fifth beta-strands of the (beta/alpha)(7)beta-barrel catalytic domain, and the OSB product is located between the Lys 133 at the end of the second beta-strand and the Lys 235 at the end of the sixth beta-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 Mg(2+).SHCHC complex of the K133R mutant has been solved at 1.62 A resolution by molecular replacement starting from the structure of the Mg(2+).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 Mg(2+).OSB, the side chain of Lys 133 is appropriately positioned to function as the only acid/base catalyst in the syn dehydration. The epsilon-ammonium group of Lys 235 forms a cation-pi 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.

• Authors: Vadim A Klenchin, Erika A Taylor Ringia, John A Gerlt, Ivan Rayment
• Journal: Biochemistry (Biochemistry) Vol. 42 Issue 49 Pg. 14427-33 (Dec 16 2003) ISSN: 0006-2960 [Print] United States
• PMID: 14661953 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• 2-succinyl-6-hydroxycyclohexa-2,4-diene-1-carboxylic acid
• Cyclohexanes
• Escherichia coli Proteins
• Salicylates
• Succinates
• Carbon-Carbon Lyases
• o-succinylbenzoic acid synthase
• Phosphopyruvate Hydratase

Topics

• Amino Acid Substitution (genetics)
• Binding Sites (genetics)
• Carbon-Carbon Lyases (chemistry, genetics, metabolism)
• Catalysis
• Circular Dichroism
• Crystallography, X-Ray
• Cyclohexanes
• Escherichia coli Proteins (chemistry, genetics, metabolism)
• Evolution, Molecular
• Kinetics
• Mutagenesis, Site-Directed
• Phosphopyruvate Hydratase (chemistry, genetics, metabolism)
• Salicylates (chemistry, metabolism)
• Succinates (chemistry, metabolism)