Biosynthesis and Purification of Nicotinamide Adenine Dinucleotide for Mechanistic Studies of Toxins

Nicotinamide adenine dinucleotide (NAD⁺) has many key biological roles, including its function as an essential co-factor as well as acting as a signaling molecule. For these reasons, dysregulation of NAD⁺ is characteristic in several key pathologies (e.g. colorectal cancer and necrotizing fasciitis). Moreover, it is not surprising that toxins such as the cholera toxin, diphtheria toxin, and tuberculosis necrotizing toxin utilize this activity to induce host cell death. Even while toxic beta-NAD glycohydrolases pose a serious threat, there is still much to be learned about their roles in pathogenesis. Only with a better understanding of how these enzymes function will it be possible to develop better diagnostic and therapeutic agents. In hopes of developing chemical biology tools to probe the role of beta-NAD⁺ glycohydrolases, NAD⁺ analogs with a wide range of pK_a values were biosynthesized and purified. With the natural substrate analogs in hand, it will be possible to better understand the enzyme catalyzed reaction, thereby providing a blueprint for future probe designs.