Author:
Gutierrez-Rus Luis I.,Alcalde Miguel,Risso Valeria A,Sanchez-Ruiz Jose M.
Abstract
AbstractThe routine generation of enzymes with completely new active sites is one of the major unsolved problems in protein engineering. Advances in this field have been so far modest, perhaps due, at least in part, to the widespread use of modern natural proteins as scaffolds for de novo engineering. Most modern proteins are highly evolved and specialized, and, consequently, difficult to repurpose for completely new functionalities. Conceivably, resurrected ancestral proteins with the biophysical properties that promote evolvability, such as high stability and conformational diversity, could provide better scaffolds for de novo enzyme generation. Kemp elimination, a non-natural reaction that provides a simple model of proton abstraction from carbon, has been extensively used as a benchmark in de novo enzyme engineering. Here, we present an engineered ancestral β-lactamase with a new active site capable of efficiently catalysing the Kemp elimination. Our Kemp eliminase is the outcome of a minimalist design based on a single function-generating mutation followed by sharply-focused, low-throughput library screening. Yet, its catalytic parameters (kcat/KM=2·105 M−1s−1, kcat=635 s−1) compare favourably with the average modern natural enzyme and with the best proton-abstraction de novo Kemp eliminases reported in the literature. General implications of our results for de novo enzyme engineering are discussed.
Publisher
Cold Spring Harbor Laboratory
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