title: Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation.
abstract: 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 β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-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 α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Buller
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Sabine
authors.initials: S
authors.lastname: Brinkmann-Chen
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: David K
authors.initials: DK
authors.lastname: Romney
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Michael
authors.initials: M
authors.lastname: Herger
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Javier
authors.initials: J
authors.lastname: Murciano-Calles
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125 frances@cheme.caltech.edu.
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D000494:Allosteric Regulation; D000596:Amino Acids / Q000378:metabolism; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D005818:Genetic Engineering; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology*; D012486:Salmonella typhimurium / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014367:Tryptophan Synthase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D008024:Ligands; D021122:Protein Subunits; D014367:Tryptophan Synthase
keywords: PLP; allostery; noncanonical amino acid; protein engineering