Download MendeleyComputational design of enone-binding proteins with catalytic activity for the Morita-Baylis-Hillman reaction.
Bjelic, S., Nivon, L.G., Celebi-Olcum, N., Kiss, G., Rosewall, C.F., Lovick, H.M., Ingalls, E.L., Gallaher, J.L., Seetharaman, J., Lew, S., Montelione, G.T., Hunt, J.F., Michael, F.E., Houk, K.N., Baker, D.(2013) ACS Chem Biol 8: 749-757
- PubMed: 23330600
- DOI: https://doi.org/10.1021/cb3006227
- Primary Citation of Related Structures:
3U26, 3UW6 - PubMed 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.
Organizational Affiliation:
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
