6Q7N

Crystal structure of BH32 alkylated with the mechanistic inhibitor 2-bromoacetophenone

Summary for 6Q7N

• Entry DOI: 10.2210/pdb6q7n/pdb
• Descriptor: BH32, 1-PHENYLETHANONE (3 entities in total)
• Functional Keywords: computationally designed enzyme, hydrolase
• Biological source: Pyrococcus horikoshii
• Total number of polymer chains: 1
• Total formula weight: 27796.77

Authors

Levy, C.W. (deposition date: 2018-12-13, release date: 2019-06-05, Last modification date: 2024-10-16)

Primary citation

Burke, A.J.,Lovelock, S.L.,Frese, A.,Crawshaw, R.,Ortmayer, M.,Dunstan, M.,Levy, C.,Green, A.P.
Design and evolution of an enzyme with a non-canonical organocatalytic mechanism.
Nature, 570:219-223, 2019

PubMed 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 N-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 N-methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. N-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.

PubMed: 31132786
DOI: 10.1038/s41586-019-1262-8

Experimental method

X-RAY DIFFRACTION (2.02 Å)