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Article: Identification of the enzymatic active site of CD38 by site-directed mutagenesis

TitleIdentification of the enzymatic active site of CD38 by site-directed mutagenesis
Authors
KeywordsSpecies Index: Aplysia
Issue Date2000
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, 2000, v. 275 n. 28, p. 21566-21571 How to Cite?
AbstractCD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca2+ messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phospate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR, hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15 fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.
Persistent Identifierhttp://hdl.handle.net/10722/132568
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorMunshi, Cen_HK
dc.contributor.authorAarhus, Ren_HK
dc.contributor.authorGraeff, Ren_HK
dc.contributor.authorWalseth, TFen_HK
dc.contributor.authorLevitt, Den_HK
dc.contributor.authorLee, HCen_HK
dc.date.accessioned2011-03-28T09:26:23Z-
dc.date.available2011-03-28T09:26:23Z-
dc.date.issued2000en_HK
dc.identifier.citationJournal Of Biological Chemistry, 2000, v. 275 n. 28, p. 21566-21571en_HK
dc.identifier.issn0021-9258en_HK
dc.identifier.urihttp://hdl.handle.net/10722/132568-
dc.description.abstractCD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca2+ messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phospate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR, hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15 fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.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.subjectSpecies Index: Aplysiaen_US
dc.titleIdentification of the enzymatic active site of CD38 by site-directed mutagenesisen_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.M909365199en_HK
dc.identifier.pmid10781610en_HK
dc.identifier.scopuseid_2-s2.0-0034647497en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0034647497&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume275en_HK
dc.identifier.issue28en_HK
dc.identifier.spage21566en_HK
dc.identifier.epage21571en_HK
dc.identifier.isiWOS:000088230600081-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridMunshi, C=7003972383en_HK
dc.identifier.scopusauthoridAarhus, R=6701339421en_HK
dc.identifier.scopusauthoridGraeff, R=7003614053en_HK
dc.identifier.scopusauthoridWalseth, TF=7005424273en_HK
dc.identifier.scopusauthoridLevitt, D=7102923276en_HK
dc.identifier.scopusauthoridLee, HC=26642959100en_HK

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