A Panel of TrpB Biocatalysts Derived from Tryptophan Synthase through the Transfer of Mutations that Mimic Allosteric Activation

Naturally occurring enzyme homologues often display highly divergent activity with non-natural substrates. Exploiting this diversity with enzymes engineered for new or altered function, however, is laborious because the engineering must be replicated for each homologue. A small set of mutations of the tryptophan synthase -subunit (TrpB) from Pyrococcus furiosus, which mimics the activation afforded by binding of the -subunit, was demonstrated to have a similar activating effect in different TrpB homologues with as little as 57% sequence identity. Kinetic and spectroscopic analyses indicate that the mutations function through the same mechanism: mimicry of -subunit binding. From these enzymes, we identified a new TrpB catalyst that displays a remarkably broad activity profile in the synthesis of 5-substituted tryptophans. This demonstrates that allosteric activation can be recapitulated throughout a protein family to explore natural sequence diversity for desirable biocatalytic transformations.