S35: Discovery and directed evolution of promiscuous enzymes

Our understanding of the early evolution of new metabolic functions is based upon anecdotal genetic and biochemical evidence. Some auxotrophies can be suppressed by over-expressing substrate ambiguous (those that catalyze the same chemical transformation on different substrates) or promiscuous (those that catalyze different transformations on similar substrates) enzymes. We systematically identified multi-functional enzymes within the Escherichia coli proteome. Of the 104 single-gene knockout strains screened, >20% of these auxotrophs were rescued by the over-expression of at least one non-cognate E. coli gene. The deleted gene and its suppressor were generally unrelated, suggesting that promiscuity is usually a product of contingency.  Here I focus upon an interesting exception to this rule.  Isochorismate synthase (MenF) is paralogous to para-aminobenzoate synthase (PabB), but has enough PabB activity to rescue a DpabB auxotroph when over-expressed under selective conditions.  We compared the tertiary structures of these paralogs and mutated two residues in the MenF active-site.  The engineered single and double-mutant proteins were expressed, purified and characterized.  Our analysis suggests that the mutations are synergistic in effect, such that the single mutations have no effect upon fitness.