Directed evolution of a (beta alpha)(8)-barrel enzyme to catalyze related reactions in two different metabolic pathways

Enzymes participating in different metabolic pathways often have similar ca talytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme fo r N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucl eotide (ProFAR) isomerase (HisA; EC 5.3.1.16) and phosphoribosylanthranilat e (PRA) isomerase (TrpF; EC 5.3.1,24), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a sim ilar (beta alpha)(8)-barrel structure. Using random mutagenesis and selecti on, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchan ge could establish TrpF activity on the HisA scaffold. These findings sugge st that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (beta alpha)(8)- barrel enzymes a re very suitable for the design of new catalytic activities.