Identification of glutamic acid 381 as a candidate active site residue of Pseudomonas aeruginosa exoenzyme S

Biochemistry
S LiuJoseph T Barbieri

Abstract

Exoenzyme S of Pseudomonas aeruginosa (ExoS) is a member of the family of bacterial ADP-ribosylating exotoxins (bAREs). Site-directed mutagenesis of glutamic acids within the catalytic domain of ExoS (termed delta N222) allowed the identification of the preferential inactivation of ADP-ribosyltransferase activity by alanine substitution of E381. The specific activity of E381A mutant was 0.02% of wild-type delta N222. Delta N222(E381A) retained the requirement of factor activating exoenzyme S (FAS) activation for the expression of ADP-ribosyltransferase activity. In contrast, E387A, E399A, and E414A mutants possessed ADP-ribosyltransferase activity similar to that of wild-type delta N222. Kinetic evaluation of E381A and two other mutants, E381D and E381S, showed that their primary defect was a lower kcat in the ADP-ribosylation of soybean trypsin inhibitor (SBTI). The Km for NAD and SBTI and activation by FAS varied 2- and 10-fold relative to delta N222. In addition, the E381 mutants possessed identical protease patterns during thrombin and trypsin digestion as delta N222, which indicated that E381 mutants had retained their overall conformation. Together, these data identify E381 as contributing to the catalytic activity of exo...Continue Reading

References

Jan 1, 1995·Infection and Immunity·S M KulichJ T Barbieri

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Citations

Jun 7, 2003·Advances in Enzyme Regulation·Jun SakuraiHideaki Tsuge
Aug 18, 2004·Nature Structural & Molecular Biology·Jianjun SunJoseph T Barbieri
Aug 10, 2000·Molecular Microbiology·K J PedersonJ T Barbieri
Nov 24, 2004·Infection and Immunity·Ciara M Shaver, Alan R Hauser
Mar 29, 2000·Journal of Bacteriology·M NagahamaJ Sakurai
Jan 12, 2013·Current Protein & Peptide Science·Marlies GalleRudi Beyaert
Jul 25, 2009·Nihon saikingaku zasshi. Japanese journal of bacteriology·Toshio Kodama
Dec 20, 2015·Acta Biochimica Et Biophysica Sinica·Yongcong TanYu-Feng Yao
Aug 8, 2006·Cellular Microbiology·Anthony W MaressoJoseph T Barbieri
Jun 17, 2003·The Journal of Biological Chemistry·Jianjun Sun, Joseph T Barbieri
Jul 15, 2004·The Journal of Biological Chemistry·Anthony W MaressoJoseph T Barbieri
Feb 20, 1999·Infection and Immunity·J RadkeJ T Barbieri
Mar 20, 1999·Infection and Immunity·A J VallisD W Frank

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