The molecular details of the processes involved in divergent evolution of "new" enzymatic functions are ill-defined. Likely starting points are either a progenitor promiscuous for the new reaction or a progenitor capable of catalyzing the new reaction following a single substitution that results from a single base change. However, the molecular (sequence) pathway by which the selective advantage provided by this protein can be improved and ultimately optimized is unclear. In the mechanistically diverse enolase superfamily, we discovered that a monofunctional progenitor could acquire the ability to catalyze a "new" reaction by a single base change: the D297G mutant of the monofunctional L-Ala-D/L-Glu epimerase (AEE) from Escherichia coli catalyzed a low level of the o-succinylbenzoate synthase (OSBS) reaction as well as a reduced level of the AEE 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]. We then discovered that the selective advantage and OSBS activity of the D297G mutant are both enhanced by the I19F substitution [Vick, J. E., Schmidt, D. M. Z., and Gerlt, J. A. (2005) Biochemistry 44, 11722-11729]. Both the D297G and I19F substitutions are positioned to alter the substrate specificity so that the substrate for the OSBS reaction is more productively positioned vis a vis the active site catalytic groups. We now report that both the selective advantage and OSBS activity of the D297G/I19F double mutant are enhanced by the R24C (one base change from the wild type Arg codon), R24W (two base changes from the wild type Arg codon and one base change from the R24C codon), and L277W (one base change from the wild type Leu codon) substitutions. The effects of the R24C and L277W mutants are "additive" in the D297G/I19F/R24C/L277W mutant. The greatest selective advantage and OSBS activity are associated with the D297G/I19F/R24W mutant. These "new" substitutions that enhance both the selective advantage and kinetic constants are positioned in the active site where they can alter the specificity, highlighting that the evolution of the "new" OSBS function can be accomplished by changes in substrate specificity.
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