National Center for
5E0K: X-ray Crystal Structure Of Tryptophan Synthase Complex From Pyrococcus Furiosus At 2.76 A
Directed evolution of the tryptophan synthase beta-subunit for stand-alone function recapitulates allosteric activation
Proc. Natl. Acad. Sci. U. S. A. (2015) 112 p.14599-14604
Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the beta-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native alphabetabetaalpha complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the alpha-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the alpha-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope.