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

Biochemistry. 2003 Dec 16;42(49):14427-33. doi: 10.1021/bi035545v.


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

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


  • 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

Associated data

  • PDB/1R6W