Characterization of the active site of ADP-ribosyl cyclase.
ADP-ribosyl cyclase synthesizes two Ca(2+) 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. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) 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 Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and that Glu(179) may indeed be the catalytic residue.
Munshi
C
C
Department of Physiology, Minneapolis, Minnesota 55455, USA.
Thiel
D J
DJ
Mathews
I I
II
Aarhus
R
R
Walseth
T F
TF
Lee
H C
HC
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000345:Affinity Labels; D019943:Amino Acid Substitution; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001048:Aplysia / Q000201:enzymology; D001386:Azides / Q000493:pharmacokinetics; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism / Q000493:pharmacokinetics; D009244:NAD+ Nucleosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010843:Pichia; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000345:Affinity Labels; D015703:Antigens, CD; D000943:Antigens, Differentiation; D001386:Azides; D011994:Recombinant Proteins; D009243:NAD; C069931:nicotinamide 8-azidoadenine dinucleotide; D004247:DNA; D036541:ADP-ribosyl Cyclase; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1