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Titolo:
Identification of the enzymatic active site of CD38 by site-directed mutagenesis
Autore:
Munshi, C; Aarhus, R; Graeff, R; Walseth, TF; Levitt, D; Lee, HC;
Indirizzi:
Univ Minnesota, Dept Pharmacol, Minneapolis, MN 55455 USA Univ Minnesota Minneapolis MN USA 55455 rmacol, Minneapolis, MN 55455 USA Univ Minnesota, Dept Physiol, Minneapolis, MN 55455 USA Univ Minnesota Minneapolis MN USA 55455 hysiol, Minneapolis, MN 55455 USA
Titolo Testata:
JOURNAL OF BIOLOGICAL CHEMISTRY
fascicolo: 28, volume: 275, anno: 2000,
pagine: 21566 - 21571
SICI:
0021-9258(20000714)275:28<21566:IOTEAS>2.0.ZU;2-9
Fonte:
ISI
Lingua:
ENG
Soggetto:
CYCLIC ADP-RIBOSE; NICOTINAMIDE ADENINE-DINUCLEOTIDE; CD8(+) T-CELLS; FUNCTIONAL EXPRESSION; ANTIGEN EXPRESSION; CRYSTAL-STRUCTURE; PICHIA-PASTORIS; CYCLASE; HYDROLYSIS; MOLECULE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
39
Recensione:
Indirizzi per estratti:
Indirizzo: Lee, HC Univ Minnesota, Dept Pharmacol, 321 Church St SE,4-145 Jackson Hall, Minneapolis, MN 55455 USA Univ Minnesota 321 Church St SE,4-145 Jackson Hall Minneapolis MN USA 55455
Citazione:
C. Munshi et al., "Identification of the enzymatic active site of CD38 by site-directed mutagenesis", J BIOL CHEM, 275(28), 2000, pp. 21566-21571

Abstract

CD38 is a ubiquitous protein originally identified as a lymphocyte antigenand 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 phosphate. 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 Glu226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gin, 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 Gin 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 observedwhen 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 resultsindicate a strong structural homology between the active sites of CD38 andthe Aplysia cyclase.

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Documento generato il 23/09/20 alle ore 09:14:22