An adenine dinucleotide variant of the nickel-pincer nucleotide cofactor in ornithine cyclodeaminases

The nickel-pincer nucleotide (NPN), which was first identified in lactate racemase, is a widespread cofactor present in around one-fifth of bacterial species and more than half of archaeal species. However, only a fraction of the enzymes utilizing this cofactor have been identified. In this study, we identified several novel enzyme families that are potentially dependent on NPN, including one that is usually linked to arginine catabolism. Four enzymes from this family, which were isolated from various genetic sources, exhibited ornithine cyclodeaminase (OCD) activity when co-expressed with the NPN biosynthetic enzymes. Biochemical analysis revealed that these enzymes utilize a unique cofactor: a nickel-pincer adenine dinucleotide (NPAD), a novel derivative of NPN. Cofactor exchange experiments and mass spectrometry confirmed NPAD as the active cofactor, which fits better in the structure of OCDs than NAD⁺. This finding expands the known roles of nickel-pincer cofactors, introducing NPAD as a critical cofactor in Bacteria, where it supports nitrogen remobilization and amino acid fermentation, and in Archaea, where it is involved in L-proline biosynthesis. This discovery not only highlights the versatility of the nickel-pincer cofactor and suggests the presence of other undiscovered enzyme families that may rely on NPAD.