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Evolutionary potential of (beta/alpha)8-barrels: in vitro enhancement of a "new" reaction in the enolase superfamily.
- Source :
-
Biochemistry [Biochemistry] 2005 Sep 06; Vol. 44 (35), pp. 11722-9. - Publication Year :
- 2005
-
Abstract
- The repertoire of reactions in the mechanistically diverse enolase superfamily is the result of divergent evolution that conserved enolization of a carboxylate anion substrate but allowed different overall reactions using different substrates. Details of the pathways for the natural evolutionary process are unknown, but the events reasonably involve (1) incremental increases in the level of the "new" reaction that would provide a selective advantage and (2) an accompanying loss of the "old" reaction catalyzed by the progenitor. In an effort to better understand the molecular processes of divergent evolution, the D297G mutant of the l-Ala-d/l-Glu epimerase (AEE) from Escherichia coli was designed so that it could bind the substrate for the o-succinylbenzoate synthase (OSBS) reaction and, as a result, catalyze that reaction [Schmidt, D. M. Z., Mundorff, E. C., Dojka, M., Bermudez, E., Ness, J. E., Govindarajan, S., Babbitt, P. C., Minshull, J., and Gerlt, J. A. (2003) Biochemistry 42, 8387-8393]. The AEE progenitor did not catalyze the OSBS reaction, but the D297G mutant catalyzed a low level of the OSBS reaction (k(cat), 0.013 s(-)(1); K(m), 1.8 mM; k(cat)/K(m), 7.4 M(-)(1) s(-)(1)) that was sufficient to permit anaerobic growth by an OSBS-deficient strain of E. coli; the level of the progenitor's natural AEE reaction was significantly diminished. Using random mutagenesis and an anaerobic metabolic selection, we now have identified the I19F substitution as an additional mutation that enhances both growth of the OSBS-deficient strain and the kinetic constants for the OSBS reaction (k(cat), 0.031 s(-)(1); K(m), 0.34 mM; k(cat)/K(m), 90 M(-)(1) s(-)(1)). Several other substitutions for Ile 19 also enhanced the level of the OSBS reaction. All of the substitutions substantially decreased the level of the AEE reaction from that possessed by the D297G progenitor. The changes in the kinetic constants for both the OSBS and AEE reactions are attributed to a readjustment of substrate specificity so that the substrate for the OSBS reaction is more productively presented to the conserved acid/base catalysts in the active site. These observations support our hypothesis that evolution of "new" functions in the enolase superfamily can occur simply by changes in specificity-determining residues.
- Subjects :
- Amino Acid Isomerases genetics
Amino Acid Isomerases metabolism
Amino Acid Sequence
Amino Acid Substitution
Bacillus subtilis enzymology
Binding Sites
Carbon-Carbon Lyases chemistry
Escherichia coli enzymology
Escherichia coli Proteins chemistry
Evolution, Molecular
Gene Library
Intramolecular Lyases genetics
Intramolecular Lyases metabolism
Mutagenesis, Site-Directed
Phosphopyruvate Hydratase genetics
Phosphopyruvate Hydratase metabolism
Protein Structure, Secondary
Racemases and Epimerases chemistry
Substrate Specificity
Carbon-Carbon Lyases metabolism
Escherichia coli Proteins genetics
Escherichia coli Proteins metabolism
Phenylbutyrates metabolism
Racemases and Epimerases genetics
Racemases and Epimerases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 44
- Issue :
- 35
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 16128573
- Full Text :
- https://doi.org/10.1021/bi050963g