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Article: A Single Residue at the Active Site of CD38 Determines Its NAD Cyclizing and Hydrolyzing Activities

TitleA Single Residue at the Active Site of CD38 Determines Its NAD Cyclizing and Hydrolyzing Activities
Authors
KeywordsChemicals And Cas Registry Numbers
Issue Date2001
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, 2001, v. 276 n. 15, p. 12169-12173 How to Cite?
AbstractCD38 is a multifunctional enzyme involved in metabolizing two Ca 2+ messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). When incubated with NAD, CD38 predominantly hydrolyzes it to ADP-ribose (NAD glycohydrolase), but a trace amount of cADPR is also produced 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, purified, 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 activity. A series of additional replacements was made at the Glu-146 position including Ala, Asn, Gly, Asp, and Leu. All the mutants exhibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly. E146A showed the highest cyclase activity, which was more than 3-fold higher than its hydrolysis activity. All mutants also cyclized nicotinamide guanine dinucleotide to produce cyclic GDP. This activity was enhanced likewise, with E146A showing more than 9-fold higher activity than the wild type. In addition to NAD, CD38 also hydrolyzed cADPR effectively, and 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, only minimally affected the base-exchange activity that is responsible for synthesizing NAADP. Homology modeling was used to assess possible structural changes at the active site of E146A. These results are consistent with Glu-146 being crucial in controlling specifically and selectively the cyclase and hydrolase activities of CD38.
Persistent Identifierhttp://hdl.handle.net/10722/132566
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorGraeff, Ren_HK
dc.contributor.authorMunshi, Cen_HK
dc.contributor.authorAarhus, Ren_HK
dc.contributor.authorJohns, Men_HK
dc.contributor.authorLee, HCen_HK
dc.date.accessioned2011-03-28T09:26:21Z-
dc.date.available2011-03-28T09:26:21Z-
dc.date.issued2001en_HK
dc.identifier.citationJournal Of Biological Chemistry, 2001, v. 276 n. 15, p. 12169-12173en_HK
dc.identifier.issn0021-9258en_HK
dc.identifier.urihttp://hdl.handle.net/10722/132566-
dc.description.abstractCD38 is a multifunctional enzyme involved in metabolizing two Ca 2+ messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). When incubated with NAD, CD38 predominantly hydrolyzes it to ADP-ribose (NAD glycohydrolase), but a trace amount of cADPR is also produced 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, purified, 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 activity. A series of additional replacements was made at the Glu-146 position including Ala, Asn, Gly, Asp, and Leu. All the mutants exhibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly. E146A showed the highest cyclase activity, which was more than 3-fold higher than its hydrolysis activity. All mutants also cyclized nicotinamide guanine dinucleotide to produce cyclic GDP. This activity was enhanced likewise, with E146A showing more than 9-fold higher activity than the wild type. In addition to NAD, CD38 also hydrolyzed cADPR effectively, and 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, only minimally affected the base-exchange activity that is responsible for synthesizing NAADP. Homology modeling was used to assess possible structural changes at the active site of E146A. These results are consistent with Glu-146 being crucial in controlling specifically and selectively the cyclase and hydrolase activities of CD38.en_HK
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_HK
dc.relation.ispartofJournal of Biological Chemistryen_HK
dc.subjectChemicals And Cas Registry Numbersen_US
dc.titleA Single Residue at the Active Site of CD38 Determines Its NAD Cyclizing and Hydrolyzing Activitiesen_HK
dc.typeArticleen_HK
dc.identifier.emailGraeff, R: graeffr@hku.hken_HK
dc.identifier.emailLee, HC: leehc@hku.hken_HK
dc.identifier.authorityGraeff, R=rp01464en_HK
dc.identifier.authorityLee, HC=rp00545en_HK
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1074/jbc.M011299200en_HK
dc.identifier.pmid11278881-
dc.identifier.scopuseid_2-s2.0-0035853770en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0035853770&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume276en_HK
dc.identifier.issue15en_HK
dc.identifier.spage12169en_HK
dc.identifier.epage12173en_HK
dc.identifier.isiWOS:000168081800099-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridGraeff, R=7003614053en_HK
dc.identifier.scopusauthoridMunshi, C=7003972383en_HK
dc.identifier.scopusauthoridAarhus, R=6701339421en_HK
dc.identifier.scopusauthoridJohns, M=35978377900en_HK
dc.identifier.scopusauthoridLee, HC=26642959100en_HK

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