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Article: Characterization of the active site of ADP-ribosyl cyclase

TitleCharacterization of the active site of ADP-ribosyl cyclase
Authors
Issue Date1999
PublisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/
Citation
Journal Of Biological Chemistry, 1999, v. 274 n. 43, p. 30770-30777 How to Cite?
AbstractADP-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.
Persistent Identifierhttp://hdl.handle.net/10722/171661
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorMunshi, Cen_US
dc.contributor.authorThiel, DJen_US
dc.contributor.authorMathews, IIen_US
dc.contributor.authorAarhus, Ren_US
dc.contributor.authorWalseth, TFen_US
dc.contributor.authorLee, HCen_US
dc.date.accessioned2012-10-30T06:16:13Z-
dc.date.available2012-10-30T06:16:13Z-
dc.date.issued1999en_US
dc.identifier.citationJournal Of Biological Chemistry, 1999, v. 274 n. 43, p. 30770-30777en_US
dc.identifier.issn0021-9258en_US
dc.identifier.urihttp://hdl.handle.net/10722/171661-
dc.description.abstractADP-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.en_US
dc.languageengen_US
dc.publisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/en_US
dc.relation.ispartofJournal of Biological Chemistryen_US
dc.subject.meshAdp-Ribosyl Cyclaseen_US
dc.subject.meshAffinity Labelsen_US
dc.subject.meshAmino Acid Substitutionen_US
dc.subject.meshAnimalsen_US
dc.subject.meshAntigens, Cden_US
dc.subject.meshAntigens, Cd38en_US
dc.subject.meshAntigens, Differentiation - Chemistry - Genetics - Metabolismen_US
dc.subject.meshAplysia - Enzymologyen_US
dc.subject.meshAzides - Pharmacokineticsen_US
dc.subject.meshBase Sequenceen_US
dc.subject.meshBinding Sitesen_US
dc.subject.meshCloning, Molecularen_US
dc.subject.meshCrystallography, X-Rayen_US
dc.subject.meshDna - Chemistry - Geneticsen_US
dc.subject.meshKineticsen_US
dc.subject.meshModels, Molecularen_US
dc.subject.meshMutagenesis, Site-Directeden_US
dc.subject.meshNad - Analogs & Derivatives - Metabolism - Pharmacokineticsen_US
dc.subject.meshNad+ Nucleosidase - Chemistry - Genetics - Metabolismen_US
dc.subject.meshPichiaen_US
dc.subject.meshProtein Conformationen_US
dc.subject.meshRecombinant Proteins - Chemistry - Metabolismen_US
dc.titleCharacterization of the active site of ADP-ribosyl cyclaseen_US
dc.typeArticleen_US
dc.identifier.emailLee, HC:leehc@hku.hken_US
dc.identifier.authorityLee, HC=rp00545en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1074/jbc.274.43.30770en_US
dc.identifier.pmid10521467-
dc.identifier.scopuseid_2-s2.0-0032719465en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0032719465&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume274en_US
dc.identifier.issue43en_US
dc.identifier.spage30770en_US
dc.identifier.epage30777en_US
dc.identifier.isiWOS:000083276700063-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridMunshi, C=7003972383en_US
dc.identifier.scopusauthoridThiel, DJ=7005794841en_US
dc.identifier.scopusauthoridMathews, II=35474611200en_US
dc.identifier.scopusauthoridAarhus, R=6701339421en_US
dc.identifier.scopusauthoridWalseth, TF=7005424273en_US
dc.identifier.scopusauthoridLee, HC=26642959100en_US

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