Nicotinamide mononucleotide adenylyltransferase (NMNAT) is an indispensable enzyme in the biosynthesis and salvage of NAD and NADP in all living organisms. In prokaryotes, it is absolutely required for cell survival, thus representing an attractive target for designing new broad-spectrum anti-infectious pharmaceutics. There is also considerable medical interest in human NMNAT because it catalyzes the key step in the metabolic conversion of the antitumor drug tiazofurin to its active form, tiazofurin adenine dinucleotide (TAD). Because of the vital roles of NAD in the cell, adequate levels of NAD must be maintained. In bacteria, a single protein NadR performs multiple functions in response to the cellular NAD and ATP levels. These include a transcriptional repressor, a NMN adenlyltransferase, and a nicotinamide ribose kinase. NadR regulates the expression of the genes involved in both de novo biosynthesis and savage of NAD. It also controls the uptake of NMN and its subsequent adenylation. Structural information of NMNAT and NadR, along with their complexes with substrates will be essential for understanding the catalysis, substrate recognition, inhibition, as well as regulation of these important enzymes. We have solved the crystal structure of the first bacterial NMNAT in its ligand free form and have obtained well diffracting human NMNAT crystals. Comparison of the bacterial and human enzyme structures will help to design specific bacterial inhibitors with high selectivity. Additionally, we have expressed and purified NadR proteins from S. typhimurium and H. influenzae, and the crystallization trials are in progress. Elucidating the structures of NadR in its various ligand-bound forms and its complex with DNA will reveal how its conformation and function are modulated by both NAD and ATP.
