Evolutionary Potential of (β/α)₈-Barrels:  In Vitro Enhancement of a “New” Reaction in the Enolase Superfamily

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.

Kinetic and Structural Characterization of a Heterohexamer 4-Oxalocrotonate Tautomerase from Chloroflexus aurantiacus J-10-fl: Implications for Functional and Structural Diversity in the Tautomerase Superfamily,

“…The improvement in activity came about by an increase in substrate specificity. This sequence of events likely mimics (on some level) nature’s process to generate new enzymatic activities, that is, a promiscuous enzyme is first created and then enhanced by a small number of mutations (39,40). The hh4-OT may likewise develop into an interesting model system to explore the evolution of CaaD activity in the tautomerase superfamily.…”

Evolutionary Optimization of Computationally Designed Enzymes: Kemp Eliminases of the KE07 Series

“…Although some directed evolution experiments resulted in N 10,000-fold increases in activity via only a small number of mutations, these cases are relatively rare (for examples, see Refs. [26][27][28]. It was also demonstrated that while the promiscuous functions of natural enzymes are plastic and can be easily altered by mutations, their native, primary functions, which had been under selection for long periods, are more robust to the effects of mutations.…”

Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily

“…Wild-type GKL exhibited promiscuous paraoxonase activity, and not surprisingly, the double mutation led to an abrogation of paraoxonase activity. In the natural evolution of enzyme function, newly acquired reactivities are often obtained at the expense of old reactions (27). The double mutant gained new substrate reactivity (toward C4-HSL) and with it, had significant decreases in catalytic efficiencies of 5.7-and 215-fold for ␥-nonalactone and ␦-nonalactone, respectively.…”