title: A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases.
abstract: The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity. This heuristic-based approach leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale. We demonstrate the efficacy of this strategy by inverting the cofactor specificity of four structurally diverse NADP-dependent enzymes: glyoxylate reductase, cinnamyl alcohol dehydrogenase, xylose reductase, and iron-containing alcohol dehydrogenase. The analytical components of this approach have been fully automated and are available in the form of an easy-to-use web tool: Cofactor Specificity Reversal-Structural Analysis and Library Design (CSR-SALAD).
authors.affiliation: Division of Chemistry and Chemical Engineering, and ‡Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91125, United States.
authors.forename: Jackson K B
authors.initials: JK
authors.lastname: Cahn
authors.affiliation: Division of Chemistry and Chemical Engineering, and ‡Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91125, United States.
authors.forename: Caroline A
authors.initials: CA
authors.lastname: Werlang
authors.affiliation: Division of Chemistry and Chemical Engineering, and ‡Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91125, United States.
authors.forename: Armin
authors.initials: A
authors.lastname: Baumschlager
authors.affiliation: Division of Chemistry and Chemical Engineering, and ‡Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91125, United States.
authors.forename: Sabine
authors.initials: S
authors.lastname: Brinkmann-Chen
authors.affiliation: Division of Chemistry and Chemical Engineering, and ‡Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91125, United States.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Mayo
authors.affiliation: Division of Chemistry and Chemical Engineering, and ‡Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91125, United States.
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000235:genetics; D000449:Aldehyde Reductase / Q000235:genetics; D009243:NAD / Q000235:genetics*; D009249:NADP / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D013379:Substrate Specificity
chemical_list: D009243:NAD; D009249:NADP; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; C018656:cinnamyl alcohol dehydrogenase; D000449:Aldehyde Reductase; C020830:glyoxylate reductase
keywords: cofactor specificity; library design; oxidoreductases; protein engineering; semirational engineering