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).

The focal point of our study was the phosphotriesterase (PTE) from ​Pseudomonas diminuta. PTE is a promiscuous metalloenzyme: in addition to highly efficient hydrolysis of the organophosphate pesticide paraoxon (​k_cat/K_M approximately 10​⁸ M​^-1​s​^-1​), it promiscuously hydrolyzes esters, lactones, and diverse organophosphates, including toxic nerve agents, such as VX, Russian VX, soman (GD), and cyclosarin (GF), albeit with k_cat/K_M values that are orders-of-magnitude lower than for paraoxon. Effective organophosphate detoxification, however, demands high catalytic efficiency, with ​k_cat/K_M of 10​⁷ M​^-1​min^​-1 considered a minimum for​ in vivo protection, thereby motivating several recent enzyme-engineering efforts that targeted PTE​. Furthermore, the growing threat from a new generation of nerve agents, similar in structure to VX and GF​​, emphasizes the need for broad-spectrum nerve-agent hydrolases. FuncLib’s goal is to design a small set of stable, efficient,and functionally diverse multipoint active-site mutants suitable for low-throughput experimental testing. The design strategy is general and can be applied, in principle, to any natural enzyme starting from its molecular structure and adiverse set of homologous sequences.

References

1. ↑ REF