Automated method of computational enzyme identification and design
Inventors
Zanghellini, Alexandre • Ban, Yih-En Andrew • ALTHOFF, Eric Anthony • Grabs, Daniela • Azoitei, Mihai Luchian
Assignees
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Publication Number
US-11393556-B2
Publication Date
2022-07-19
Expiration Date
Abstract
The invention provides computational methods for engineering, selecting, and/or identifying proteins with a desired activity. Further provided are automated computational design and screening methods to engineer proteins with desired functional activities including, but not limited to ligand binding, catalytic activity, substrate specificity, regioselectivity and/or stereoselectivity.
Core Innovation
The invention relates to a method for making a protein having an enzymatic activity by starting from a template structure of a template protein having the enzymatic activity. In the described method, the template protein is a dehydrogenase, and a functional site description is prepared based on the template structure. The functional site description includes amino acid identities for each functional site amino acid residue, rotameric states for each functional residue, rotameric states for each ligand, and geometric placement of the functional residues with respect to said ligand.
The method computationally selects amino acid sequences that have structural homology and/or sequence homology to the template protein from a database of amino acid sequences. For each computationally selected sequence, a structural model is provided, and at least one amino acid sequence is then selected based on evaluation. The evaluation includes computationally docking ligand(s), substrate(s), transition state(s), or reaction product(s) relating to the enzymatic activity.
Finally, the selected sequence(s) are recombinantly expressed and enzymatic activity is confirmed, thereby making the protein having the enzymatic activity. The enzymatic activity is specifically dehydrogenase activity, tying the computational functional-site definition, homology-based selection/modeling, and docking-based evaluation to experimental confirmation of dehydrogenase function.
Claims Coverage
Only one independent claim is provided. It combines a template dehydrogenase structure, a functional site description including rotameric states and geometric placement relative to ligand, computational selection of homologous sequences, structural modeling, docking-based evaluation using ligand/substrate/transition-state/reaction-product entities, and recombinant expression with confirmation of dehydrogenase enzymatic activity.
Template dehydrogenase structure and functional site description
Obtaining a template structure of a template protein having the enzymatic activity wherein the template protein is a dehydrogenase, and preparing a functional site description based on the template structure, wherein the functional site description comprises amino acid identities for each functional site amino acid residue, rotameric states for each functional residue, rotameric states for each ligand, and geometric placement of the functional residues with respect to said ligand.
Homology-based sequence selection and structural modeling
Computationally selecting, from a database of amino acid sequences, one or more amino acid sequences having structural homology and/or sequence homology to the template protein, and providing a structural model for each of the computationally selected one or more amino acid sequences.
Docking-based evaluation using enzymatic activity entities
Selecting, based on an evaluation of the computationally selected one or more amino acid sequences, at least one amino acid sequence that satisfies the prepared functional site description, the evaluation including computationally docking ligand(s), substrate(s), transition state(s), or reaction product(s) relating to the enzymatic activity.
Recombinant expression with confirmation of dehydrogenase activity
Recombinantly expressing and confirming the enzymatic activity for one or more of the selected at least one amino acid sequence, thereby making the protein having the enzymatic activity, wherein the enzymatic activity is dehydrogenase activity.
The claim coverage centers on a dehydrogenase-focused protein engineering method that connects a template-based functional-site description to homology-based sequence selection and structural modeling, followed by docking-based evaluation and recombinant expression with confirmation of dehydrogenase enzymatic activity.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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