Mechanism of cyclizing NAD to cyclic ADP-ribose by ADP-ribosyl cyclase and CD38.
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.
Graeff
Richard
R
Department of Physiology, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Liu
Qun
Q
Kriksunov
Irina A
IA
Kotaka
Masayo
M
Oppenheimer
Norman
N
Hao
Quan
Q
Lee
Hon Cheung
HC
D051997:ADP-ribosyl Cyclase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D055162:Biocatalysis; D002118:Calcium / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D036563:Cyclic ADP-Ribose / Q000378:metabolism*; D003500:Cyclization; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009243:NAD / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D013379:Substrate Specificity
D009243:NAD; D036563:Cyclic ADP-Ribose; D051997:ADP-ribosyl Cyclase 1; D002118:Calcium