Structural Basis for Enzymatic Evolution from a Dedicated ADP-ribosyl Cyclase to a Multifunctional NAD Hydrolase

Cyclic ADP-ribose (cADPR) is a universal calcium messenger molecule that regulates many physiological processes. The production and degradation of cADPR are catalyzed by a family of related enzymes, including the ADP-ribosyl cyclase from Aplysia california (ADPRAC) and CD38 from human. Although ADPRC and CD38 share a common evolutionary ancestor, their enzymatic functions toward NAD and cADPR homeostasis have evolved divergently. Thus, ADPRC can only generate cADPR from NAD (cyclase), whereas CD38, in contrast, has multiple activities, i.e. in cADPR production and degradation, as well as NAD hydrolysis (NADase). In this study, we determined a number of ADPRC and CD38 structures bound with various nucleotides. From these complexes, we elucidated the structural features required for the cyclization (cyclase) reaction of ADPRC and the NADase reaction of CD38. Using the structural approach in combination with site-directed mutagenesis, we identified Phe-174 in ADPRC as a critical residue in directing the folding of the substrate during the cyclization reaction. Thus, a point mutation of Phe-174 to glycine can turn ADPRC from a cyclase toward an NADase. The equivalent residue in CD38, Thr-221, is shown to disfavor the cyclizing folding of the substrate, resulting in NADase being the dominant activity. The comprehensive structural comparison of CD38 and APDRC presented in this study thus provides insights into the structural determinants for the functional evolution from a cyclase to a hydrolase.Cyclic ADP-ribose (cADPR)³ is a calcium messenger ubiquitous in mammals as well as in invertebrates and plants and is responsible for regulating many physiological processes ranging from the simple function of calcium channel operation to the complex higher level organization of hormone secretion and autism (reviewed in Lee (1), Schuber and Lund (2), and Malavasi et al. (3)). The enzymatic production of cADPR from the substrate nicotinamide adenine dinucleotide (NAD) requires first the removal of the nicotinamide moiety followed by a cyclization reaction in which both ends of the remaining nucleotide are annealed (Fig. 1A). ADP-ribosyl cyclase (ADPRC) from Aplysia california was the first enzyme discovered to possess this function (cyclase) (4). Based on sequence homology (5), two human antigens, CD38 and CD157, were identified to also have the cyclase activity (6–8). However, different from ADPRC, which produces only cADPR from NAD, CD38/CD157 has evolved more like an NADase, producing mainly ADP-ribose (ADPR) from NAD, with cADPR being a minor product. The acquisition of the NADase and the cADPR hydrolysis activities of CD38 make it an important signaling enzyme in regulating NAD and cADPR homeostasis (9–11). Genetic analysis, as well as the conservation of sequence and disulfide bonds among these enzymes, establish that they all evolved from a common ancestor (12). Little is known of why this conserved family of enzymes has evolved divergently in their catalytic metabolism of NAD and cADPR.Open in a separate windowFIGURE 1.Schemes of cADPR formation and mechanistic analogs for substrate and product. A, the cyclization reaction producing cADPR from NAD is catalyzed by both ADPRC and CD38. The structural difference between cADPR and N1-cIDPR lies at the 6-position of purine ring (6-NH for cADPR; 6-O for N1-cIDPR). B, an analog of the substrate NAD, N(2F-A)D, is enzymatically converted to 2F-ADPR by ADPRC instead of cyclized to c(2F-A)DPR. The formation of cADPR from NAD requires the intramolecular attack of the reaction intermediate by the adenine N1 atom. The addition of a fluorine atom on the adjacent C2 atom of adenine prevents the cyclization from occurring. C, ara-2′F-NAD and ribo-2′F-NAD are analogs of NAD that inhibit the cyclization reaction by producing covalent adducts during the catalysis by CD38. Both analogs differ only in the orientation of their fluorine atoms at the 2′-position of the adenine ribose.ADPRC, however, is not solely a cyclase because it can also catalyze the hydrolysis of NMN into ribose-5-phosphate and nicotinamide (13, 14). The catalytic outcome of this novel enzyme is thus determined not by the enzyme alone but also by the specific interactions between the active site and a particular substrate. Consistently, using an NAD analog, N(2F-A)D, as substrate, Zhang et al. (15) showed that the hydrolase activity of ADPRC can be dominantly revealed, whereas its cyclase activity is suppressed beyond detection (Fig. 1B). Likewise, human CD38 can be converted to a ADPRC-like enzyme by mutation of a single residue, Glu-146, at the active site (16). In this study, we determined the structural determinants critical for the catalytic characteristics of ADPRC and CD38 by comparing the crystal structures of the complexes of ADPRC and CD38 bound with various catalytically revealing substrates and products (Fig. 1, A–C). The results identify residues Phe-174 in the cyclase and Thr-221 in CD38 as the main determinants for the cyclase and hydrolysis activities of the enzymes. All together, these structures provide insights into the structural requirements for functional evolution from a cyclase to a hydrolase.