-10 6 M -1 s -1 for TrpF activity. Not only are there naturally occurring bifunctional enzymes, but promiscuous TrpF activity has been detected on both ancestral and extant specialist HisA enzymes, with kcat values ranging from 10 -4 to 10 -2 s -1 . HisA has also been converted into TrpF by directed evolution, and in serial passaging experiments. In the latter study, laboratory evolution of the ProFAR-specific HisA (lacking TrpF activity) from Salmonella enterica over 3,000 generations yielded an extensive suite of mutations in the S. enterica HisA (SeHisA) that resulted in specialist HisA enzymes, specialist TrpF enzymes and PriA-like bifunctional enzymes. green, yellow and dark red on each structure, respectively. Loop 1 in TmTrpF is short (four residues), which is why no corresponding loop is annotated on this panel. Note that for clarity, N7D and A176D reversions were applied to the structure of SeHisA in complex with ProFAR (these reversions were also applied in our simulations, as described in the Methodology section). (D) The proposed mechanism for the Amadori rearrangement leading to the isomerization of substrates ProFAR and PRA by the different enzymes. As shown in Figure , HisA and PriA are decorated by three long catalytic loops, loops 1, 5, and 6 (or two analogous loops in the case of TrpF, which has lost most of loop 1). Of these loops, loop 5 carries key residues that are important for substrate binding, loop 6 carries the catalytically important aspartic acid side chain, and a number of mutations important for interaction with substrate PRA have been observed at position 15 of loop 1. The mutants generated by Näsvall et al. were the subject of detailed structural and biochemical analyses. As with PriA, this analysis of the evolved bifunctional SeHisA variants indicated that the bifunctionality is driven by competition between not just the substrates ProFAR and PRA, but also between structurally distinct conformations of loops 1 and 5, in particular 28 (Figure ).