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

Ashleigh J. Burke, Sarah L. Lovelock, Amina Frese, Rebecca Crawshaw, Mary Ortmayer, Mark Dunstan, Colin Levy and Anthony P. Green ()
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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

Nature, 2019, vol. 570, issue 7760, 219-223

Abstract: Abstract The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1–4. 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 organocatalysis5, 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 design6–10. 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 dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.

Date: 2019
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DOI: 10.1038/s41586-019-1262-8

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