Efficient Base-Catalyzed Kemp Elimination in an Engineered Ancestral Enzyme

Abstract

The routine generation of enzymes with completely new active sites is a major unsolved problem in protein engineering. Advances in this field have thus far been 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 that is capable of efficiently catalyzing Kemp elimination. The engineering of our Kemp eliminase involved minimalist design based on a single function-generating mutation, inclusion of an extra polypeptide segment at a position close to the de novo active site, and sharply focused, low-throughput library screening. Nevertheless, its catalytic parameters (kcat/KM~2·105 M−1 s−1, kcat~635 s−1) compare favorably with the average modern natural enzyme and match the best proton-abstraction de novo Kemp eliminases that are reported in the literature. The general implications of our results for de novo enzyme engineering are discussed.

Fishing for Catalysis: Experimental Approaches to Narrowing Search Space in Directed Evolution of Enzymes

“…The best Precambrian variant GNCA4-W229D/F290W catalyzes Kemp elimination with a catalytic efficiency of 5000 M –1 s –1 , which subsequently was improved by 4-fold to 20,000 M –1 s –1 using FuncLib . Further improvement of this approach yielded a highly active enzyme with a catalytic efficiency of 200,000 M –1 s –1 …”

“… 47 Further improvement of this approach yielded a highly active enzyme with a catalytic efficiency of 200,000 M –1 s –1 . 48 …”

Protection of Catalytic Cofactors by Polypeptides as a Driver for the Emergence of Primordial Enzymes

“…It seems reasonable, therefore, that there are efficient mechanisms for the de novo emergence of completely new enzymes. Such mechanisms, however, remain elusive to protein engineers, who have found the generation of efficient de novo enzymes in the lab to be extremely challenging ( Blomberg et al 2013 ; Risso et al 2017 ; Donnelly et al 2018 ; Lovelock et al 2022 ; Gutierrez-Rus et al 2022 ; Yeh et al 2023 ). Our limited grasp of the evolutionary mechanisms of de novo enzyme generation implies a serious gap in our understanding of the origin of life.…”

Substrate Positioning Dynamics Involves a Non-Electrostatic Component to Mediate Catalysis

“…Three reasons support this choice. First, computational benchmarks and biochemical assay protocols have been established for KE07-R7-2, ensuring accuracy and reproducibility. Second, kinetic parameters have been experimentally characterized by Bhowmick et al for KE07-R7-2 mutants, providing reference kinetic data for this study.…”