title: Computational design of enone-binding proteins with catalytic activity for the Morita-Baylis-Hillman reaction.
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.
authors: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors: Sinisa
authors: S
authors: Bjelic
authors: Lucas G
authors: LG
authors: Nivón
authors: Nihan
authors: N
authors: Çelebi-Ölçüm
authors: Gert
authors: G
authors: Kiss
authors: Carolyn F
authors: CF
authors: Rosewall
authors: Helena M
authors: HM
authors: Lovick
authors: Erica L
authors: EL
authors: Ingalls
authors: Jasmine Lynn
authors: JL
authors: Gallaher
authors: Jayaraman
authors: J
authors: Seetharaman
authors: Scott
authors: S
authors: Lew
authors: Gaetano Thomas
authors: GT
authors: Montelione
authors: John Francis
authors: JF
authors: Hunt
authors: Forrest Edwin
authors: FE
authors: Michael
authors: K N
authors: KN
authors: Houk
authors: David
authors: D
authors: Baker
mesh_terms: D002384:Catalysis; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D011506:Proteins