Laboratory reconstruction of the evolution of a bi-substrate specific sugar isomerase

In histidine and tryptophan biosynthesis, two chemically equivalent isomerisation reactions are catalyzed by the (βα)₈-barrel enzymes HisA (ProFAR isomerase) and TrpF (PRA isomerase). However, some members of the Actinbacteria phylum lack a TrpF protein and instead contain the bi-specific isomerase PriA. PriA is highly similar to HisA but is able to catalyze the isomerisation of both ProFAR and PRA. A plausible evolutionary scenario is that PriA has evolved from HisA by the acquisition of few amino acid exchanges, which has made TrpF dispensable for Actinbacteria.

We wish to reconstruct the evolution of HisA into PriA in the laboratory by i) site-directed mutagenesis based on known crystal structures of the two enzymes (“rational protein design”) and ii) random mutagenesis of the hisA gene, followed by in vivo selection for a bi-specific enzyme using an Escherichia coli strain which lacks the genes for both HisA and TrpF (“library selection”). Along these lines, we have introduced an amino acid exchange into HisA from Thermotoga maritima which does not compromise its native function and at the same time leads to measurable TrpF activity. Strikingly the introduced amino acid is found in a number of naturally occurring HisA enzymes, which also show promiscuous TrpF activity. The latter activity could be increased by additional amino acid exchanges that were identified by library selection.