辅因子
NAD+激酶
代谢工程
醇脱氢酶
生物化学
计算生物学
还原酶
酶
蛋白质工程
醛还原酶
脱氢酶
化学
计算机科学
立体化学
生物
作者
John W. Cahn,Caroline Werlang,Armin Baumschlager,Sabine Brinkmann‐Chen,Stephen L. Mayo,Frances H. Arnold
标识
DOI:10.1021/acssynbio.6b00188
摘要
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).
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