This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Glutamine?dependent NAD+ synthetase catalyzes the last step of the de novo and recycling pathways in NAD+ biosynthesis. This enzyme is essential for survival of replicating and non-replicating Mycobacterium tuberculosis and as such it is an attractive drug target. In humans NAD+ synthetase independent pathways participate in NAD+ biosynthesis. We are elucidating the mechanism of regulation of the catalytic activities at active sites that are spatially separated, determining structural and mechanistic differences between the H. sapiens and M. tuberculosis enzymes and testing both kinetically and structurally inhibitors, resulting in the design of inhibitors specific for NAD+ synthetaseTB. The structure of wild type NAD+ synthetaseTB in complexes with DON and NaAD, previously solved in our lab, is a homoctameric and features a 40 ? long inter-subunit ammomia tunnel for transport of ammonia from glutaminase active site to the synthetase active site. We have been solved four structures of NAD+ synthetaseTB with different ligand bounds at range of 2.6-3.0 ? resolutions. In our attempt to obtain a structure in complexes with ligands that stabilized active site loops P2 at the synthetase active site. We hypothesize that ordering of loop P2 induces a conformational change that activates the glutaminase active site. The crystals were co-crystallized in 1.4-1.8 M ammonium citrate as wild type does. The structure complexes showed the binding of ATP or AMP and NaAD at the synthetase active site but Mg2+ or PPi/Mg2+ were not found. Mg2+ ions were found to stabilize loop P2 by coordinating with ATP or NaAD-AMP intermediate and the use of high ammonia citrate at high concentration as a precipitant may interfere with the coordination mediated by Mg2+ ions, resulting in loop P2 were still disordered in any structures solved. Now, we have several crystals with analog of NaAD-AMP and PPi at the synthetase active site (10 rod-shaped, 2 plate-like, and 4 pyramid-shaped crystals), which were crystallized in three brand new crystallization conditions. These crystals may provide the different space group with structural information of loop P2 and the activated conformation. In addition, we are currently working on the human enzyme, which we can get expressed in Sf9 insect cell line. Crystals of native enzyme with analog of NaAD-AMP and PPi were collected at the SSRL syntchrostron at 3.0 ? resolution and we are solving structure using molecular replacement. If it fails due to the low sequence homology of the human NAD synthetase with search model we will need to generate heavy-atom derivatives and solve the structure by SAD/MAD.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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Brookhaven National Laboratory
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