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Regulation of active site coupling in glutamine-dependent NAD+ synthetase

Abstract

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.

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Figure 1: The reactions catalyzed at the synthetase and glutaminase domains of NAD+ synthetaseGln.
Figure 2: Oligomeric assembly of NAD+ synthetaseGln from M. tuberculosis.
Figure 3: DON inhibition of the glutaminase domain.
Figure 4: The homooctameric structure of NAD+ synthetaseGln with all eight intersubunit tunnels.
Figure 5: The ammonia tunnel and the synthetase active site.
Figure 6: Connecting elements between glutaminase and synthetase active sites.

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Acknowledgements

We thank L.-Y. Gao (University of Maryland) and C.D. Lima (Sloan Kettering Institute) for M. tuberculosis genomic DNA and for the expression plasmids pSMT3 and Ulp1, respectively. We thank K. Rajashankar for assistance with data collection and processing. We thank A. Wlodawer and, especially, J.D. Kahn for critiques on the manuscript. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines of the Advanced Photon Source, supported by award RR-15301 from the National Center for Research Resources at the US National Institutes of Health. Use of the Advanced Photon Source is supported by the US Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. This work was supported by a PRF grant from the American Chemical Society to B.G. and by start-up funds to B.G. and N.L.-L. from the Department of Chemistry and Biochemistry, University of Maryland, College Park.

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M.R. cloned, expressed and purified all the proteins and performed all the kinetic experiments; N.L.-L. collected and analyzed crystallographic data and built and refined the structural model of NAD+ synthetase; B.G. designed all the experiments, crystallized native and SeMet NAD+ synthetase and contributed to the building and the refinement of the structural model of NAD+ synthetase; B.G. and N.L.-L. wrote the manuscript.

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Correspondence to Nicole LaRonde-LeBlanc or Barbara Gerratana.

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LaRonde-LeBlanc, N., Resto, M. & Gerratana, B. Regulation of active site coupling in glutamine-dependent NAD+ synthetase. Nat Struct Mol Biol 16, 421–429 (2009). https://doi.org/10.1038/nsmb.1567

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