A single residue at the active site of CD38 determines its NAD cyclizing and hydrolyzing activities

CD38 is a multifunctional enzyme involved in metabolizing two Ca2+ messenge rs, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosp hate (NAADP). When incubated with NAD, CD38 predominantly hydrolyzes it to ADP-ribose (NAD glycohydrolase), but a trace amount of cADPR is also produc ed through cyclization of the substrate. Site-directed mutagenesis was used to investigate the amino acid important for controlling the hydrolysis and cyclization reactions. CD38 and its mutants were produced in yeast, purifi ed, and characterized by immunoblot, Glu-146 is a conserved residue present in the active site of CD38. Its replacement with Phe greatly enhanced the cyclization activity to a level similar to that of the NAD hydrolysis activ ity. A series of additional replacements was made at the Glu-146 position i ncluding Ala, Asn, Gly, Asp, and Leu. All the mutants exhibited enhanced cy clase activity to various degrees, whereas the hydrolysis activity was inhi bited greatly. E146A showed the highest cyclase activity, which was more th an 3-fold higher than its hydrolysis activity. All mutants also cyclized ni cotinamide guanine dinucleotide to produce cyclic GDP. This activity was en hanced likewise, with E146A showing more than g-fold higher activity than t he wild type. In addition to NAD, CD38 also hydrolyzed cADPR effectively, a nd this activity was correspondingly depressed in the mutants. When all the mutants were considered, the two cyclase activities and the two hydrolase activities were correlated linearly. The Glu-146 replacements, however, onl y minimally affected the base-exchange activity that is responsible for syn thesizing NAADP. Homology modeling was used to assess possible structural c hanges at the active site of E146A. These results are consistent with Glu-1 46 being crucial in controlling specifically and selectively the cyclase an d hydrolase activities of CD38.