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. 1995 Aug;63(8):3182–3186. doi: 10.1128/iai.63.8.3182-3186.1995

Functional domains of Pseudomonas aeruginosa exoenzyme S.

D A Knight 1, V Finck-Barbançon 1, S M Kulich 1, J T Barbieri 1
PMCID: PMC173434  PMID: 7622246

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.

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Selected References

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