Journal:Molecular Cell:2

Automated design of efficient and functionally diverse enzyme repertoires

Olga Khersonsky, Rosalie Lipsh, Ziv Avizemer, Yacov Ashani, Moshe Goldsmith, Haim Leader, Orly Dym, Shelly Rogotner, Devin L. Trudeau, Jaime Prilusky, Pep Amengual-Rigo, Victor Guallar, Dan S. Tawfik, and Sarel J. Fleishman ^[1]

Molecular Tour
Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) effects on activity. We describe FuncLib - an automated method to design multipoint mutants at enzyme active sites using phylogenetic analysis and Rosetta design calculations. FuncLib was applied to two unrelated enzymes, a phosphotriesterase (PTE) and an acetyl-CoA synthetase. All designs were active, and most showed activity profiles that significantly differed from wild type and from one another. Several dozen designs with only 3-6 active-site mutations nevertheless exhibited 10-4,000-fold higher efficiencies with a range of alternative substrates, including the hydrolys is of the toxic nerve agents soman and cyclosarin and the synthesis of butyryl-CoA - activities that are hardly detectable in the wild type enzymes. FuncLib designs included epistatic active-site mutations that are unlikely to be accessible to natural and laboratory evolution; the method circumvents high-throughput screens and opens the way to design highly efficient and diverse catalytic repertoires. FuncLib is implemented as a web-server (http://funclib.weizmann.ac.il). References

1. ↑ REF