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Simultaneous optimization of enzyme activity and quaternary structure by directed evolution
Author(s) -
Vamvaca Katherina,
Butz Maren,
Walter Kai U.,
Taylor Sean V.,
Hilvert Donald
Publication year - 2005
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.051431605
Subject(s) - random hexamer , dna shuffling , directed evolution , protein quaternary structure , trimer , protein engineering , shuffling , directed molecular evolution , enzyme , chorismate mutase , chemistry , biology , computational biology , biochemistry , mutant , gene , computer science , dimer , protein subunit , programming language , biosynthesis , organic chemistry
Natural evolution has produced efficient enzymes of enormous structural diversity. We imitated this natural process in the laboratory to augment the efficiency of an engineered chorismate mutase with low activity and an unusual hexameric topology. By applying two rounds of DNA shuffling and genetic selection, we obtained a 400‐fold more efficient enzyme, containing three non‐active‐site mutations. Detailed biophysical characterization of the evolved variant suggests that it exists predominantly as a trimer in solution, but is otherwise similarly stable as the parent hexamer. The dramatic structural and functional effects achieved by a small number of seemingly innocuous substitutions highlights the utility of directed evolution for modifying protein–protein interactions to produce novel quaternary states with optimized activities.