期刊
ACS SYNTHETIC BIOLOGY
卷 6, 期 2, 页码 326-333出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.6b00188
关键词
cofactor specificity; oxidoreductases; protein engineering; library design; semirational engineering
资金
- Gordon and Betty Moore Foundation [GBMF2809]
- American Recovery and Reinvestment Act (ARRA) funds through the National Institutes of Health Shared Instrumentation Grant Program [S10RR027203]
- Resnick Sustainability Institute (Caltech)
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: glyoxyiate 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).
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