Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes

Background: Directed evolution by DNA shuffling has been used to modify phy sical and catalytic properties of biological systems. We have shuffled two highly homologous triazine hydrolases and conducted an exploration of the s ubstrate specificities of the resulting enzymes to acquire a better underst anding of the possible distributions of novel functions in sequence space. Results: Both parental enzymes and a library of 1600 variant triazine hydro lases were screened against a synthetic library of 15 triazines. The shuffl ed library contained enzymes with up to 150-fold greater transformation rat es than either parent. It also contained enzymes that hydrolyzed five of ei ght triazines that were not substrates for either starting enzyme. Conclusions: Permutation of nine amino acid differences resulted in a set o f enzymes with surprisingly diverse patterns of reactions catalyzed. The fu nctional richness of this small area of sequence space may aid our understa nding of both natural and artificial evolution. (C) 2001 Elsevier Science L td. All rights reserved.