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Design and evolution of an enzyme with a non-canonical organocatalytic mechanism

DOI: 10.1038/s41586-019-1262-8 DOI Help

Authors: Ashleigh J. Burke (University of Manchester) , Sarah L. Lovelock (University of Manchester) , Amina Frese (University of Manchester) , Rebecca Crawshaw (University of Manchester) , Mary Ortmayer (University of Manchester) , Mark Dunstan (University of Manchester) , Colin Levy (University of Manchester) , Anthony P. Green (University of Manchester)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature , VOL 453

State: Published (Approved)
Published: May 2019
Diamond Proposal Number(s): 12788 , 17773

Abstract: The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.

Journal Keywords: Biocatalysis; Hydrolases; Protein design

Subject Areas: Chemistry


Instruments: I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography