Publications

• Evolution of enzymatic activity in the enolase superfamily: functional studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis.

  Taylor Ringia E.A., Garrett J.B., Thoden J.B., Holden H.M., Rayment I., Gerlt J.A.

  o-Succinylbenzoate synthase (OSBS) from Amycolatopsis, a member of the enolase superfamily, catalyzes the Mn2+-dependent exergonic dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate (SHCHC) to 4-(2'-carboxylphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB) in the menaquinone bi ... >> More

  o-Succinylbenzoate synthase (OSBS) from Amycolatopsis, a member of the enolase superfamily, catalyzes the Mn2+-dependent exergonic dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate (SHCHC) to 4-(2'-carboxylphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB) in the menaquinone biosynthetic pathway. This enzyme first was identified as an N-acylamino acid racemase (NAAAR), with the optimal substrates being the enantiomers of N-acetyl methionine. This laboratory subsequently discovered that this protein is a much better catalyst of the OSBS reaction, with the value of k(cat)/K(M), for dehydration, 2.5 x 10(5) M(-1) s(-1), greatly exceeding that for 1,1-proton transfer using the enantiomers of N-acetylmethionine as substrate, 3.1 x 10(2) M(-1) s(-1) [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-8]. The efficiency of the promiscuous NAAAR reaction is enhanced with alternate substrates whose structures mimic that of the SHCHC substrate for the OSBS reaction, for example, the value of k(cat)/K(M) for the enantiomers of N-succinyl phenylglycine, 2.0 x 10(5) M(-1) s(-1), is comparable to that for the OSBS reaction. The mechanisms of the NAAAR and OSBS reactions have been explored using mutants of Lys 163 and Lys 263 (K163A/R/S and K263A/R/S), the putative acid/base catalysts identified by sequence alignments with other OSBSs, including the structurally characterized OSBS from Escherichia coli. Although none of the mutants display detectable OSBS or NAAAR activities, K163R and K163S catalyze stereospecific exchange of the alpha-hydrogen of N-succinyl-(S)-phenylglycine with solvent hydrogen, and K263R and K263 catalyze the stereospecific exchange the alpha-hydrogen of N-succinyl-(R)-phenylglycine, consistent with formation of a Mn2+-stabilized enolate anion intermediate. The rates of the exchange reactions catalyzed by the wild-type enzyme exceed those for racemization. That this enzyme can catalyze two different reactions, each involving a stabilized enediolate anion intermediate, supports the hypothesis that evolution of function in the enolase superfamily proceeds by pathways involving functional promiscuity. << Less

  Biochemistry 43:224-229(2004) [PubMed] [EuropePMC]

• Menaquinone (vitamin K2) biosynthesis: cloning, nucleotide sequence, and expression of the menC gene from Escherichia coli.

  Sharma V., Meganathan R., Hudspeth M.E.S.

  The benzenoid aromatic compound o-succinylbenzoic acid is formed by dehydration of the prearomatic compound 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid by the enzyme o-succinylbenzoate synthase, encoded by the menC gene. A 1.3-kb PstI-PvuII fragment was found to complement the menC m ... >> More

  The benzenoid aromatic compound o-succinylbenzoic acid is formed by dehydration of the prearomatic compound 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid by the enzyme o-succinylbenzoate synthase, encoded by the menC gene. A 1.3-kb PstI-PvuII fragment was found to complement the menC mutation. The complete nucleotide sequence of this fragment revealed a single open reading frame of 954 bp capable of encoding a 35-kDa protein. A consensus sequence for a ribosomal binding site but no promoter consensus sequences were found. However, the first base of the initiating codon of this open reading frame overlaps the upstream menB gene termination codon, suggesting an operon-like organization for these genes. Consistent with this suggestion, the menB promoter can initiate transcription of the menC gene. << Less

  J. Bacteriol. 175:4917-4921(1993) [PubMed] [EuropePMC]

• Unexpected divergence of enzyme function and sequence: 'N-acylamino acid racemase' is o-succinylbenzoate synthase.

  Palmer D.R., Garrett J.B., Sharma V., Meganathan R., Babbitt P.C., Gerlt J.A.

  A protein identified as "N-acylamino acid racemase" from Amycolaptosis sp. is an inefficient enzyme (kcat/Km = 3.7 x 10(2) M-1 s-1). Its sequence is 43% identical to that of an unidentified protein encoded by the Bacillus subtilis genome. Both proteins efficiently catalyze the o-succinylbenzoate s ... >> More

  A protein identified as "N-acylamino acid racemase" from Amycolaptosis sp. is an inefficient enzyme (kcat/Km = 3.7 x 10(2) M-1 s-1). Its sequence is 43% identical to that of an unidentified protein encoded by the Bacillus subtilis genome. Both proteins efficiently catalyze the o-succinylbenzoate synthase reaction in menaquinone biosynthesis (kcat/Km = 2.5 x 10(5) and 7.5 x 10(5) M-1 s-1, respectively), suggesting that this is their "correct" metabolic function. Their membership in the mechanistically diverse enolase superfamily provides an explanation for the catalytic promiscuity of the protein from Amycolaptosis. The adventitious promiscuity may provide an example of a protein poised for evolution of a new enzymatic function in the enolase superfamily. This study demonstrates that the correct assignment of function to new proteins in functional and structural genomics may require an understanding of the metabolism of the organism. << Less

  Biochemistry 38:4252-4258(1999) [PubMed] [EuropePMC]

• 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.

  Klenchin V.A., Taylor Ringia E.A., Gerlt J.A., Rayment I.

  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-oxobutyra ... >> More

  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. << Less

  Biochemistry 42:14427-14433(2003) [PubMed] [EuropePMC]

• Evolution of enzymatic activity in the enolase superfamily: structure of o-succinylbenzoate synthase from Escherichia coli in complex with Mg(2+) and o-Succinylbenzoate.

  Thompson T.B., Garrett J.B., Taylor E.A., Meganathan R., Gerlt J.A., Rayment I.

  The X-ray structures of the ligand free (apo) and the Mg(2+)*o-succinylbenzoate (OSB) product complex of o-succinylbenzoate synthase (OSBS) from Escherichia coli have been solved to 1.65 and 1.77 A resolution, respectively. The structure of apo OSBS was solved by multiple isomorphous replacement i ... >> More

  The X-ray structures of the ligand free (apo) and the Mg(2+)*o-succinylbenzoate (OSB) product complex of o-succinylbenzoate synthase (OSBS) from Escherichia coli have been solved to 1.65 and 1.77 A resolution, respectively. The structure of apo OSBS was solved by multiple isomorphous replacement in space group P2(1)2(1)2(1); the structure of the complex with Mg(2+)*OSB was solved by molecular replacement in space group P2(1)2(1)2. The two domain fold found for OSBS is similar to those found for other members of the enolase superfamily: a mixed alpha/beta capping domain formed from segments at the N- and C-termini of the polypeptide and a larger (beta/alpha)(7)beta barrel domain. Two regions of disorder were found in the structure of apo OSBS: (i) the loop between the first two beta-strands in the alpha/beta domain; and (ii) the first sheet-helix pair in the barrel domain. These regions are ordered in the product complex with Mg(2+)*OSB. As expected, the Mg(2+)*OSB pair is bound at the C-terminal end of the barrel domain. The electron density for the phenyl succinate component of the product is well-defined; 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. The loop between the first two beta-strands in the alpha/beta motif interacts with the aromatic ring of OSB. Lys(133) and Lys(235) are positioned to function as acid/base catalysts in the dehydration reaction. Few hydrogen bonding or electrostatic interactions are involved in the binding of OSB to the active site; instead, most of the interactions between OSB and the protein are either indirect via water molecules or via hydrophobic interactions. As a result, evolution of both the shape and the volume of the active site should be subject to few structural constraints. This would provide a structural strategy for the evolution of new catalytic activities in homologues of OSBS and a likely explanation for how the OSBS from Amycolaptosis also can 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) Biochemistry 38, 4252-4258]. << Less

  Biochemistry 39:10662-10676(2000) [PubMed] [EuropePMC]