Acidic residues at the active sites of CD38 and ADP-ribosylt cyclase determine nicotinic acid adenine dinucleotide phosphate (NAADP) synthesis and hydrolysis activities

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a novel metabolite of NADP that has now been established as a Ca 2+ messenger in many cellular systems. Its synthesis is catalyzed by multifunctional enzymes, CD38 and ADP-ribosyl cyclase (cyclase). The degradation pathway for NAADP is unknown and no enzyme that can specifically hydrolyze it has yet been identified. Here we show that CD38 can, in fact, hydrolyze NAADP to ADP-ribose 2′-phosphate. This activity was low at neutrality but greatly increased at acidic pH. This novel pH dependence suggests that the hydrolysis is determined by acidic residues at the active site. X-ray crystallography of the complex of CD38 with one of its substrates, NMN, showed that the nicotinamide moiety was in close contact with Glu 146 at 3.27 Å and Asp 155 at 2.52Å. Changing Glu 146 to uncharged Gly and Ala, and Asp 155 to Gln and Asn, by site-directed mutagenesis indeed eliminated the strong pH dependence. Changing Asp 155 to Glu, in contrast, preserved the dependence. The specificity of the two acidic residues was further demonstrated by changing the adjacent Asp 147 to Val, which had minimal effect on the pH dependence. Crystallography confirmed that Asp147 was situated and directed away from the bound substrate. Synthesis of NAADP catalyzed by CD38 is known to have strong preference for acidic pH, suggesting that Glu 146 and Asp 155 are also critical determinants. This was shown to be case by mutagensis. Likewise, using similar approaches, Glu 98 of the cyclase, which is equivalent to Glu 146 in CD38, was found to be responsible for controlling the pH dependence of NAADP synthesis by the cyclase. Based on these findings, a catalytic model is proposed. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.