title: Functional domains of Pseudomonas aeruginosa exoenzyme S.
abstract: Recombinant exoenzyme S (rHisExoS) of Pseudomonas aeruginosa was expressed in Escherichia coli as a soluble, cytosolic His fusion protein. rHisExoS was purified by Ni(2+)-affinity chromatography in the presence of protease inhibitors without detectable degradation. rHisExoS possessed a specific activity (within twofold) for the factor-activating exoenzyme S-dependent ADP-ribosylation of soybean trypsin inhibitor (SBTI) similar to that of native exoenzyme S. Analysis of several deletion peptides showed that delta N222, which encoded the carboxyl-terminal 222 amino acids of exoenzyme S, possessed factor-activating exoenzyme S-dependent ADP-ribosyltransferase activity. delta N222 catalyzed the ADP-ribosylation of SBTI at a rate sixfold greater than rHisExoS. Relative to rHisExoS, delta N222 had a similar affinity for NAD, a threefold greater affinity for SBTI, and a four- to eightfold greater kcat for the ADP-ribosylation of SBTI. Like native exoenzyme S, rHisExoS chromatographed as an aggregate with an apparent molecular mass of > 300 kDa. In contrast, delta N222 did not chromatograph as an aggregate, which showed that the amino-terminal 99 amino acids of exoenzyme S were responsible for the aggregation phenotype.
authors.affiliation: Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Knight
authors.forename: V
authors.initials: V
authors.lastname: Finck-Barbançon
authors.forename: S M
authors.initials: SM
authors.lastname: Kulich
authors.forename: J T
authors.initials: JT
authors.lastname: Barbieri
mesh_terms: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D003001:Cloning, Molecular; D007700:Kinetics; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014361:Trypsin Inhibitors / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D014361:Trypsin Inhibitors; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C444820:exoenzyme S