Directed evolution of the tryptophan synthase [Beta]-subunit for stand-alone function recapitulates allosteric activation

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 evolut...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 112; no. 47; p. 14599
Main Authors Buller, Andrew R, Brinkmann-Chen, Sabine, Romney, David K, Herger, Michael, Murciano-Calles, Javier, Arnold, Frances H
Format Journal Article
LanguageEnglish
Published Washington National Academy of Sciences 24.11.2015
Subjects
Amino acids
Chemical reactions
Enzymes
Evolution
Kinetics
Microorganisms
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Summary: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 ...-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native ... complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the ...-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 ...-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope. (ProQuest: ... denotes formulae/symbols omitted.)
ISSN:0027-8424
1091-6490