Enzymatic synthesis and characterization of nicotinamide riboside pseudouridine diphosphate

Nicotinamide adenine dinucleotide (NAD) analogs are of great interests for chemical and biological applications. In this work, a novel NAD analog, nicotinamide riboside pseudouridine diphosphate (NpUD) was synthesized via coupling of pseudouridine triphosphate and nicotinamide mononucleotide catalyzed by a mutant of nicotinic acid mononucleotide adenylyltransferase. It was found that NpUD is more stable than NAD against a common pyrophosphatase NudC found in Escherichia coli. Interestingly, NpUD could function as redox cofactor for several oxidoreductases, among them a mutant methanol dehydrogenase showed 1.9-fold higher activity with NpUD than that with NAD. Overall, the results offer opportunities to explore NpUD in other applications.