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Characterization of the Active Site of ADP-ribosyl Cyclase*

C. Munshi,D. Thiel,I. Mathews,R. Aarhus,T. Walseth,Hon-cheung Lee

Published 1999 in Journal of Biological Chemistry

ABSTRACT

ADP-ribosyl cyclase synthesizes two Ca2+ messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu98, Asn107 and Trp140 were within 3.5 Å of the bound nicotinamide and appeared to coordinate it. Substituting Glu98 with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16–222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2–3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu179 with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp77 to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [32P]8-azido-NAD. These results indicate that both Glu179 and Trp77 are crucial for catalysis and that Glu179 may indeed be the catalytic residue.

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