Enzyme Nicotinamide Cofactor Specificity Reversal Guided by Automated Structural Analysis and Library Design

The specificity of enzymes for nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as redox carriers can pose a significant hurdle for metabolic engineering and synthetic biology applications, where switching the specificity might be beneficial. We have developed an easy-to-use computational tool (CSR-SALAD) for the design of mutant libraries to simplify the process of reversing the cofactor specificity of an enzyme. Here, we describe the optimal use of this tool and present methods for its application in a laboratory setting.