This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Substrate channeling is defined as the transfer of a metabolite from an active site to another without exchanging in the solvent. Although in several cases substrate channeling is controversial and far from being settled, it has been experimentally implicated in the reactions catalyzed by the ubiquitous multidomain enzymes of the glutamine amidotransferase (GAT) superfamily. These enzymes catalyze key metabolic reactions in protein, amino acid, cofactor, purine and pyrimidine biosyntheses, all of which have in common the hydrolysis of glutamine to glutamate and ammonia in the glutaminase active site. The ammonia produced is transfered through an inter-domain tunnel (the ammonia tunnel) to a second active site in the acceptor domain (a synthase or synthetase domain). NAD+ synthetase represents an unprecented and uncharacterized class of the GAT superfamily. It catalyzes the ATP-dependent transformation of nicotinic acid adenine dinucleotide (NaAD+) to NAD+ , the last step of the de novo biosynthesis of NAD+. NAD+ is historically known as the cofactor of enzymes involved in reduction-oxidation reactions. An emerging and important role for NAD+ is as source of molecules involved in cell signaling, cell division, cell longevity and immune response. While in humans NAD+ is obtained either from de novo biosynthesis or from the pyridine nucleotide recycling pathways, in M. tuberculosis NAD+ production relies entirely on the de novo biosynthetic pathway. The main goal of this project is to establish a link between catalysis and local dynamics in the protein and to characterize the mechanisms of interdomain signaling and of ammonia transfer in NAD+ synthetase. This project will contribute to the basic knowledge of substrate channeling and enzyme catalysis.