Application of a fluorometric assay for characterization of the catalytic competency of a domain III fragment of Pseudomonas aeruginosa exotoxin A

Analytical Biochemistry
Souzan Armstrong, A Rod Merrill

Abstract

Pseudomonas aeruginosa exotoxin A (ETA) is a member of the family of bacterial ADP-ribosylating toxins that use NAD(+) as the ADP-ribose donor. The reaction catalyzed by ETA involves the nucleophilic attack of the diphthamide residue on the anomeric carbon of the nicotinamide ribose forming a new glycosidic bond. A fluorometric assay involving the use of etheno-beta-nicotinamide adenine dinucleotide (epsilon-NAD(+)), an analog of NAD(+), has been found to provide a rapid, reliable, and sensitive procedure for assessing the kinetic parameters of this class of enzymes including ETA and its C-terminal fragment, PE24. Furthermore, application of this new assay facilitated the determination of the kinetic parameters for the protein substrate of ETA, elongation factor, which has previously been difficult to characterize. These findings provide new insights into catalytic mechanism of dipthamide-specific ribosyltransferases. In addition, this assay should also prove valuable for the study of NADases or NAD(+)-glycohydrolase enzymes (B. Weng, W. C. Thompson, H. J. Kim, R. L. Levine, and J. Moss, 1999, J. Biol. Chem. 274, 31797-31803; Y. S. Cho, M. K. Han, O. S. Kwark, M. S. Phoe, Y. S. Cha, N. H. An, and U. H. Kim, 1998, Comp. Physiol....Continue Reading

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Citations

Apr 27, 2012·The Journal of Biological Chemistry·Robert J FieldhouseA Rod Merrill
Sep 13, 2012·The Journal of Biological Chemistry·Adin ShnifferA Rod Merrill
Sep 21, 2012·The Journal of Biological Chemistry·Danielle VisschedykA Rod Merrill
Sep 26, 2008·Trends in Biochemical Sciences·Robert J Fieldhouse, A Rod Merrill
Jan 13, 2006·Trends in Biochemical Sciences·Susan P YatesA Rod Merrill
Sep 10, 2015·Biochemistry·Ravikiran RavulapalliA Rod Merrill
Feb 16, 2008·The Journal of Biological Chemistry·René JørgensenA Rod Merrill
Jul 5, 2006·The Biochemical Journal·Chun KimStefan H E Kaufmann
Aug 23, 2007·Clinical Chemistry·Christopher BachranHendrik Fuchs
Jul 18, 2001·The Journal of Biological Chemistry·S P Yates, A R Merrill
Feb 7, 2016·Journal of Biomolecular Screening·Ana Filipa PintoHerwig Schüler
Mar 24, 2016·The Journal of Biological Chemistry·Bronwyn LyonsA Rod Merrill
Dec 6, 2014·The Journal of Biological Chemistry·Daniel KrskaA Rod Merrill
May 28, 2019·International Journal of Biological Macromolecules·Zeinab Amiri TehranizadehMohammad Reza Saberi
Jul 14, 2019·Comparative Immunology, Microbiology and Infectious Diseases·A Prisilla, P Chellapandi

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