Enzyme catalysts for diels-alder reactions
AssigneeUniversity of Washington
InventorsDavid Baker, Alexandre Zangellini, Justin Siegel, Jennifer St. Clair
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Abstract
The present invention provides enzyme catalysts for Diels-Alder reactions, including intermolecular Diels-Alder reactions, as well as protein scaffolds for making such enzyme catalysts. In other aspects, the invention provides methods of making the enzyme catalysts, including by de novo computational design. The present invention thereby provides enzyme catalysts capable of catalyzing a desired Diels-Alder reaction, including with a specified or desired stereo-selectivity.
Claims26
A method for making an enzyme that catalyzes a Diels-Alder reaction, the method comprising:
- creating a Diels-Alder active site model comprising a diene component, a dienophile component, and hydrogen bond donor and hydrogen bond acceptor amino acid functional groups positioned to support catalysis;
- placing the Diels-Alder active site model in a Diels-Alder scaffold selected from Loligo vulgaris diisopropylfluorophosphatase, Pseudomonas putida ketosteroid isomerase, or a sequence homolog or structural homolog thereof having a 6-bladed beta propeller fold or an alpha+beta roll fold;
- introducing mutations in the scaffold to prepare an active site having the protein functional groups in position to stabilize the Diels-Alder transition state, and having a surface complementary to the Diels-Alder transition state;
- introducing mutations in the scaffold as guided by computational design and Diels-Alder assay;
- and
- expressing the Diels-Alder enzyme and testing for the Diels-Alder enzyme activity.
A method for making an enzyme that catalyzes a Diels-Alder reaction, the method comprising:
- providing a Diels-Alder scaffold having at least 30% sequence identity to SEQ ID NO:
- 2 or SEQ ID NO:9, and having a 6-bladed beta propeller fold or an alpha+beta roll fold,
- introducing one or more mutations in the Diels-Alder scaffold, and
- contacting the Diels-Alder scaffold with desired Diels-Alder substrate, and quantifying Diels-Alder enzyme activity to thereby create a Diels-Alder enzyme,
- expressing the Diels-Alder enzyme.
Dependencies are inferred from the claim text.