NAD⁺ is an essential metabolite both as a cofactor in energy metabolism and redox homeostasis and as a regulator of cellular processes. In contrast to humans, Mycobacterium tuberculosis NAD⁺ biosynthesis is absolutely dependent on the activity of a multifunctional glutamine-dependent NAD⁺ synthetase, which catalyzes the ATP-dependent formation of NAD⁺ at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. Here we report the kinetics and structural characterization of M. tuberculosis NAD⁺ synthetase. The kinetics data strongly suggest tightly coupled regulation of the catalytic activities. The structure, the first of a glutamine-dependent NAD⁺ synthetase, reveals a homooctameric subunit organization suggesting a tight dependence of catalysis on the quaternary structure, a 40-Å intersubunit ammonia tunnel and structural elements that may be involved in the transfer of information between catalytic sites.