@misc{10481/64102,
year = {2020},
month = {6},
url = {http://hdl.handle.net/10481/64102},
abstract = {Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library
screening remains sluggish, in large part due to futile probing of mutations that are catalytically neutral
and/or impair stability and folding. FuncLib is a novel approach which uses phylogenetic analysis and
Rosetta design to rank enzyme variants with multiple mutations, on the basis of predicted stability. Here,
we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The
similarity between the Michaelis complex and transition state for the enzymatic reaction makes this
system particularly challenging to optimize. Yet, experimental screening of a small number of active-site
variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers
( 2   104 M 1 s 1 and  102 s 1) for this anthropogenic reaction that compare favorably to those of
modern natural enzymes. This result illustrates the promise of FuncLib as a powerful tool with which to
speed up directed evolution, even on scaffolds that were not originally evolved for those functions, by
guiding screening to regions of the sequence space that encode stable and catalytically diverse
enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the
optimized eliminases to within  2 kcal mol 1 and indicate that the enhanced activity is linked to better
geometric preorganization of the active site. This raises the possibility of further enhancing the stabilityguidance
of FuncLib by computational predictions of catalytic activity, as a generalized approach for
computational enzyme design},
organization = {Knut and Alice Wallenberg Foundation (Wallenberg Academy Fellowship) 	
2018.0140},
organization = {Human Frontier Science Program

RGP0041/2017},
organization = {FEDER Funds/Spanish Ministry of Science, Innovation and Universities 	
BIO2015-66426-R
RTI2018-097142-B-100},
organization = {FEDER/Junta de Andalucia - Consejeria de Economia y Conocimiento 	
E.FQM.113.UGR18},
organization = {Swedish National Infrastructure for computing (SNAC) 	
2018/2-3
2019/2-1},
publisher = {Royal Society Chemistry},
keywords = {Molecular dynamics},
keywords = {Directed evolution},
keywords = {Kemp elimination},
keywords = {Efficient catalysis},
keywords = {Potential Functions},
keywords = {Force field},
keywords = {Free energy},
keywords = {Design},
keywords = {Proteins},
keywords = {Optimization},
title = {Enhancing a de novo enzyme activity by computationally-focused ultra-low-throughput screening},
doi = {10.1039/d0sc01935f},
author = {Risso, Valeria Alejandra and Gutiérrez Rus, Luis Ignacio and Ortega Muñoz, Mariano and Santoyo González, Francisco and Gavira Gallardo, José Antonio and Sánchez Ruiz, José Manuel},
}