Mechanism of cyclizing NAD to cyclic ADP-ribose by ADP-ribosyl cyclase and CD38

Abstract

Mammalian CD38 and its Aplysia homolog, ADP-ribosyl cyclase (cyclase), are two prominent enzymes that catalyze the synthesis and hydrolysis of cyclic ADP-ribose (cADPR), a Ca(2+) messenger molecule responsible for regulating a wide range of cellular functions. Although both use NAD as a substrate, the cyclase produces cADPR, whereas CD38 produces mainly ADP-ribose (ADPR). To elucidate the catalytic differences and the mechanism of cyclizing NAD, the crystal structure of a stable complex of the cyclase with an NAD analog, ribosyl-2'F-2'deoxynicotinamide adenine dinucleotide (ribo-2'-F-NAD), was determined. The results show that the analog was a substrate of the cyclase and that during the reaction, the nicotinamide group was released and a stable intermediate was formed. The terminal ribosyl unit at one end of the intermediate formed a close linkage with the catalytic residue (Glu-179), whereas the adenine ring at the other end stacked closely with Phe-174, suggesting that the latter residue is likely to be responsible for folding the linear substrate so that the two ends can be cyclized. Mutating Phe-174 indeed reduced cADPR production but enhanced ADPR production, converting the cyclase to be more CD38-like. Changing the equivalent residue in CD38, Thr-221 to Phe, correspondingly enhanced cADPR production, and the double mutation, Thr-221 to Phe and Glu-146 to Ala, effectively converted CD38 to a cyclase. This study provides the first detailed evidence of the cyclization process and demonstrates the feasibility of engineering the reactivity of the enzymes by mutation, setting the stage for the development of tools to manipulate cADPR metabolism in vivo.

FIGURE 1.

Crystal structure of the complex of cyclase with ribo-2′F-NAD. a, chemical structure of the substrate ribo-2′F-NAD and the reactions catalyzed by CD38 and the cylase. b, crystal structure of the cyclase dimer with the intermediates at each of the active sites of the monomer. The color scheme for the secondary structures is: red, α-helix; yellow, β-sheet; gray, coil. The color scheme for the residues is: cyan, Tyr-81; beige, Phe-174; blue, Glu-179; magenta, Glu-98; purple, Phe-175. The intermediates are colored by their elements: green, carbon; red, oxygen; orange, phosphorus; blue, nitrogen; light green, fluorine. c, stereo view of the folded conformation with electron density from an omit F_o − F_c map contoured at 2.7 σ and shown as blue wire mesh. Other color schemes are the same as in b. The average B-factor is 76 Ų for the folded intermediate.

FIGURE 2.

Two conformations of the intermediate from ribo-2′F-NAD. a, the extended conformation. The orange dashed line shows that the distance between the adenine and Tyr-81 is 4.0 Å. Phe-175 is colored in purple. Other color schemes are as in Fig. 1b. b, the folded conformation. The magenta dashed line shows that the distance between the adenine and Phe-174 is 4.1 Å. c, surface view of the active site pocket with the two conformations of intermediate superimposed. The folded conformation is colored by its elements as in Fig 1a. The extended conformation is colored beige. Surface color is as follows: deep blue, Phe-174; cyan, Tyr-81; red, Glu-179; white, all other residues. d, rotational conversion between the two conformations. The folded conformation is brightly colored by its elements as in Fig. 1a. The distance between N1 of the adenine is 5.2 Å from Glu-179 in this conformation. The extended conformation is lightly colored by its elements.

FIGURE 3.

HPLC analyses of the enzymatic products from NAD produced by the cyclase and its mutants. WT cyclase (upper panel, 0.1 μg/ml), F174T (middle panel, 1 μg/ml), or F175T (lower panel, 1 μg/ml) was incubated with 100 μm NAD for the indicated times and analyzed by HPLC. The part of the HPLC chromatogram that includes only the cADPR and ADPR peaks is shown for clarity. Of the three proteins, only the F174T mutant produces ADPR. Ab, absorbance.

FIGURE 4.

HPLC analyses of the enzymatic products from NAD produced by CD38 and its mutants. WT CD38 (upper panel, 0.2 μg/ml), T221F (middle panel, 5 μg/ml), or T221F/E146A (lower panel, 2 μg/ml) was incubated with 100 μm NAD for the indicated times and analyzed by HPLC. There was no detectable cADPR production by WT CD38 under these conditions, the T221F mutant produced some detectable cADPR, and the T221F/E146A double mutant produced predominantly cADPR. Ab, absorbance.