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

Abstract

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.

• Authors: Erika A Taylor Ringia, James B Garrett, James B Thoden, Hazel M Holden, Ivan Rayment, John A Gerlt
• Journal: Biochemistry (Biochemistry) Vol. 43 Issue 1 Pg. 224-9 (Jan 13 2004) ISSN: 0006-2960 [Print] United States
• PMID: 14705949 (Publication Type: Comparative Study, Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Bacterial Proteins
• Escherichia coli Proteins
• Solvents
• Hydrogen
• Carbon-Carbon Lyases
• o-succinylbenzoic acid synthase
• Phosphopyruvate Hydratase
• Amino Acid Isomerases

Topics

• Actinomycetales (enzymology, genetics)
• Amino Acid Isomerases (chemistry, genetics)
• Bacterial Proteins (chemistry, genetics)
• Binding Sites (genetics)
• Carbon-Carbon Lyases (chemistry, genetics)
• Catalysis
• Enzyme Activation (genetics)
• Escherichia coli Proteins (chemistry, genetics)
• Evolution, Molecular
• Hydrogen (chemistry)
• Hydrogen-Ion Concentration
• Kinetics
• Mutagenesis, Site-Directed
• Phosphopyruvate Hydratase (chemistry, genetics)
• Sequence Homology, Amino Acid
• Solvents
• Substrate Specificity (genetics)