Abstract
Bovine CD38/NAD+glycohydrolase (bCD38) catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2′-fluorinated analogs of NAD+. Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1′ of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2′-OH of the substrate NAD+. Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism.
Highlights
Mammalian NAD+glycohydrolases (NADases; EC 3.3.2.5 and 3.2.2.6) catalyze the hydrolytic cleavage of the nicotinamide-ribose bond of NAD(P)+
Architecture of bovine CD38 As reported previously, we have expressed in Pichia pastoris a recombinant form of bovine CD38/NAD+glycohydrolase truncated for the first 31 amino acids that encompass the transmembrane and short intracellular domains [20]
In contrast with the other known mammalian CD38, such as human CD38 (hCD38) which contains four N-glycosylation sites [7], Bovine CD38/NAD+glycohydrolase (bCD38) is a mono-glycosylated protein at position Asn201 and its only other sequon at Asn268 is unoccupied [20]
Summary
Mammalian NAD+glycohydrolases (NADases; EC 3.3.2.5 and 3.2.2.6) catalyze the hydrolytic cleavage of the nicotinamide-ribose bond of NAD(P)+. Preponderantly described as ecto-enzymes [4], some NADases were found in intracellular compartments of various tissues/cells and a role in NAD+ salvage pathways was tentatively ascribed to this class of enzymes This situation experienced an unexpected paradigmic shift with the discovery, in invertebrates, by the group of H.C. Lee, of cyclic ADP-ribose (cADPR), a new calcium mobilizing messenger [5], and of ADPribosyl cyclase, a soluble enzyme able to convert NAD+ quasiexclusively into this cyclic metabolite [6]. Simplifying our perception of these different enzymes, our group has subsequently shown that the much studied ‘classical’ bovine NADase was able to catalyze, like CD38, ADP-ribosyl cyclase (,2% of reaction products) and cyclic ADP-ribose hydrolase reactions [11,12]. The world of the classical mammalian NAD+glycohydrolases merged with that of CD38 [14]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
