Evolutionary Potential of (β/α)₈-Barrels:  Functional Promiscuity Produced by Single Substitutions in the Enolase Superfamily

Abstract

The members of the mechanistically diverse, (beta/alpha)(8)-barrel fold-containing enolase superfamily evolved from a common progenitor but catalyze different reactions using a conserved partial reaction. The molecular pathway for natural divergent evolution of function in the superfamily is unknown. We have identified single-site mutants of the (beta/alpha)(8)-barrel domains in both the l-Ala-d/l-Glu epimerase from Escherichia coli (AEE) and the muconate lactonizing enzyme II from Pseudomonas sp. P51 (MLE II) that catalyze the o-succinylbenzoate synthase (OSBS) reaction as well as the wild-type reaction. These enzymes are members of the MLE subgroup of the superfamily, share conserved lysines on opposite sides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms. A comparison of the structures of AEE and the OSBS from E. coli was used to design the D297G mutant of AEE; the E323G mutant of MLE II was isolated from directed evolution experiments. Although neither wild-type enzyme catalyzes the OSBS reaction, both mutants complement an E. coli OSBS auxotroph and have measurable levels of OSBS activity. The analogous mutations in the D297G mutant of AEE and the E323G mutant of MLE II are each located at the end of the eighth beta-strand of the (beta/alpha)(8)-barrel and alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction. The substitutions relax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reaction with the assistance of the active site lysines. The generation of functionally promiscuous and mechanistically diverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution of enzymatic activities and also highlights the utility of the (beta/alpha)(8)-barrel as a scaffold for new function.

Engineering Monolignol 4-O-Methyltransferases to Modulate Lignin Biosynthesis

“…3D). Many examples of directed evolution have demonstrated that enzymes with promiscuous functions exhibit high plasticity; they might well be used as "generalists" to further tailor particular catalytic properties (33)(34)(35). One of our triple mutants, T133L/L139Q/E165F, catalytically prefers the guaiacyl lignin precursor, coniferyl alcohol, over sinapyl alcohol; its binding affinity for the former is about 2-fold higher than for the latter (see Table 1).…”

Establishing wild-type levels of catalytic activity on natural and artificial (βα) 8 -barrel protein scaffolds

“…As a consequence, residues important for activity can be exchanged without compromising stability. For this reason, (␤␣) 8 -barrels provide an ideal scaffold for enzyme design, and are an excellent tool to study the mechanisms of molecular evolution (10)(11)(12).…”

Phenotypic Constraints and Phenotypic Hitchhiking in a Promiscuous Enzyme

“…Typically, genetic studies of trait covariation compare complex morphological or physiological traits of multicellular organisms [20, 21, 27-29, 44-47, 72]. To our knowledge, analyses of trait covariation have not yet been extensively applied to much simpler, molecular systems like individual proteins, although measurements of more than one activity in modest numbers of proteins are not unusual in protein engineering [18,49,[73][74][75][76][77]. The advantage that molecular systems like proteins have over organisms in the wild is that they can be easily manipulated in the laboratory through mutagenesis and different kinds of selection pressure.…”

“…Available evidence for such trade-offs in proteins typically involve few (as little as two) mutant proteins and their activity on two different substrates [18,[73][74][75][76][77]. If one mutant has a high activity on substrate A and a low activity on substrate B, whereas another mutant shows the opposite relationship, a trade-off may exist.…”