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.
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.
Klenchin
Vadim A
VA
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53705, USA.
Taylor Ringia
Erika A
EA
Gerlt
John A
JA
Rayment
Ivan
I
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D019755:Carbon-Carbon Lyases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003510:Cyclohexanes; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012459:Salicylates / Q000737:chemistry / Q000378:metabolism; D013386:Succinates / Q000737:chemistry / Q000378:metabolism
C087010:2-succinyl-6-hydroxycyclohexa-2,4-diene-1-carboxylic acid; D003510:Cyclohexanes; D029968:Escherichia coli Proteins; D012459:Salicylates; D013386:Succinates; D019755:Carbon-Carbon Lyases; C052986:o-succinylbenzoic acid synthase; D010751:Phosphopyruvate Hydratase