Computational Design of
Enone-Binding Proteins with
Catalytic Activity for the Morita–Baylis–Hillman Reaction
- Publication date
- Publisher
Abstract
The Morita–Baylis–Hillman reaction forms
a carbon–carbon
bond between the α-carbon of a conjugated carbonyl compound
and a carbon electrophile. The reaction mechanism involves Michael
addition of a nucleophile catalyst at the carbonyl β-carbon,
followed by bond formation with the electrophile and catalyst disassociation
to release the product. We used Rosetta to design 48 proteins containing
active sites predicted to carry out this mechanism, of which two show
catalytic activity by mass spectrometry (MS). Substrate labeling measured
by MS and site-directed mutagenesis experiments show that the designed
active-site residues are responsible for activity, although rate acceleration
over background is modest. To characterize the designed proteins,
we developed a fluorescence-based screen for intermediate formation
in cell lysates, carried out microsecond molecular dynamics simulations,
and solved X-ray crystal structures. These data indicate a partially
formed active site and suggest several clear avenues for designing
more active catalysts