A Short Practical Guide to the Quantitative Analysis of Engineered Enzymes

Bayer, Christopher D.; Hollfelder, Florian

doi:10.1201/b19951-4

Cited by 1 publication

(2 citation statements)

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“…The possibility that slow conformational changes (on the microseconds to seconds timescale) play roles in enzyme catalytic cycles has been proposed and explored 17 . Consequently, we used NMR relaxation determinations (see Methods section and Supplementary Methods ) on the β-lactamase encoded by the most probabilistic sequence at the GNCA node (GNCA MP β-lactamase) to guide our design of a new active site.…”

Section: Resultsmentioning

confidence: 99%

“…Both proteins appear very rigid on the ps-ns timescale with an order parameter of S 2 ∼0.9 for all residues in both cases. There is, however, a clear difference on the timescale (μs-ms) commonly associated with biological processes such as substrate binding, allosteric regulation and catalytic cycle 17 . Specifically, the number of residues for which the relaxation rates cannot be explained without including a conformational exchange contribution ( R ex ) is 26 for GNCA MP β-lactamase versus 12 for TEM-1 β-lactamase ( Fig.…”

Section: Methodsmentioning

confidence: 99%

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De novo active sites for resurrected Precambrian enzymes

et al. 2017

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Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%