Computational Design of Enone-Binding Proteins with Catalytic Activity for the Morita鈥揃aylis鈥揌illman Reaction

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• 作者：Sinisa Bjelic ; Lucas G. Niv贸n ; Nihan 脟elebi-脰l莽眉m ; Gert Kiss ; Carolyn F. Rosewall ; Helena M. Lovick ; Erica L. Ingalls ; Jasmine Lynn Gallaher ; Jayaraman Seetharaman ; Scott Lew ; Gaetano Thomas Montelione ; John Francis Hunt ; Forrest Edwin Michael ; K. N. Houk ; David Baker
• 刊名：ACS Chemical Biology
• 出版年：2013
• 出版时间：April 19, 2013
• 年：2013
• 卷：8
• 期：4
• 页码：749-757
• 全文大小：442K
• 年卷期：v.8,no.4(April 19, 2013)
• ISSN：1554-8937

文摘

The Morita鈥揃aylis鈥揌illman reaction forms a carbon鈥揷arbon bond between the 伪-carbon of a conjugated carbonyl compound and a carbon electrophile. The reaction mechanism involves Michael addition of a nucleophile catalyst at the carbonyl 尾-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.