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Published February 17, 2017 | Supplemental Material + Accepted Version
Journal Article Open

A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases


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).

Additional Information

© 2017 American Chemical Society. Received: July 7, 2016; Published: September 20, 2016. This work was supported by the Gordon and Betty Moore Foundation through Grant No. GBMF2809 to the Caltech Programmable Molecular Technology Initiative and by American Recovery and Reinvestment Act (ARRA) funds through the National Institutes of Health Shared Instrumentation Grant Program, Grant No. S10RR027203, to F.H.A. J.K.B.C. acknowledges the support of the Resnick Sustainability Institute (Caltech). The authors thank Ruchi Jahagirdar and Lisa Mears for experimental assistance and Tilman Flock for providing the list of nonredundant PDBs used for CSR-SALAD testing and validation. They also thank numerous former and current members of the Arnold and Mayo laboratories for invaluable suggestions and discussions. Accession Codes: PDB: 4XDY, PDB: 4TSK, PDB: 3DOJ, PDB: 1PIW, PDB: 1K8C, PDB: 1VHD, PDB: 3PEF Author Contributions: This project was conceived by J.K.B.C., S.B-C., S.L.M., and F.H.A. J.K.B.C. designed and programmed CSR-SALAD, which was implemented online by C.A.W. J.K.B.C. performed the validation experiments with assistance from A.B., and produced the figures and tables. J.K.B.C., S.B-C., and F.H.A. wrote the manuscript with input from all authors. The authors declare no competing financial interest.

Attached Files

Accepted Version - nihms-1754172.pdf

Supplemental Material - sb6b00188_si_001.pdf


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