Abstract
NAD+ synthetase is an essential enzyme of de novo and recycling pathways of NAD+ biosynthesis in Mycobacterium tuberculosis but not in humans. This bifunctional enzyme couples the NAD+ synthetase and glutaminase activities through an ammonia tunnel but free ammonia is also a substrate. Here we show that the Homo sapiens NAD+ synthetase (hsNadE) lacks substrate specificity for glutamine over ammonia and displays a modest activation of the glutaminase domain compared to tbNadE. We report the crystal structures of hsNadE and NAD+ synthetase from M. tuberculosis (tbNadE) with synthetase intermediate analogues. Based on the observed exclusive arrangements of the domains and of the intra- or inter-subunit tunnels we propose a model for the inter-domain communication mechanism for the regulation of glutamine-dependent activity and NH3 transport. The structural and mechanistic comparison herein reported between hsNadE and tbNadE provides also a starting point for future efforts in the development of anti-TB drugs.
Highlights
NAD+ synthetase is an essential enzyme of de novo and recycling pathways of NAD+ biosynthesis in Mycobacterium tuberculosis but not in humans
NAD+ is the cofactor of enzymes involved in reduction–oxidation reactions but other important roles for it have emerged in recent years in cell signalling, cell division, cell longevity, immune response and cancer[1,2,3,4,5,6], and as a possible target in pathogenic bacteria[7,8] such as Mycobacterium tuberculosis (Mtb)[9]
The few kinetic and structural studies of glnNAD+ synthetases have shown that these enzymes are complex multifunctional enzymes displaying catalytic coupling between two reactions[22,23,26,27]
Summary
NAD+ synthetase is an essential enzyme of de novo and recycling pathways of NAD+ biosynthesis in Mycobacterium tuberculosis but not in humans. We report the structural and kinetic characterizations of hsNadE and a direct structural comparison of the closed and active conformation of (1) hsNadE structure in complex with a synthetase intermediate analog at 2.84 Å resolution and (2) tbNadE structure in complex with glutamine, sulfonamide derivative 1 (5′-O-(N-(nicotinyl)sulfamoyl) adenosine; SFI), and PPi at 3.14 Å resolution. Both structures are snapshots of the enzymes in the allosteric state with the synthetase active site loop ordered revealing an exclusive rearrangement of the glutaminase active site and the molecular tunnels. The kinetic and structural characterizations of hsNadE enable a direct comparison between these two homologs to develop and rationally design inhibitors that selectively inhibit tbNadE
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