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 Ni2+-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 ΔN222, which encoded the carboxyl-terminal 222 amino acids of exoenzyme S, possessed factor-activating exoenzyme S-dependent ADP-ribosyltransferase activity. ΔN222 catalyzed the ADP-ribosylation of SBTI at a rate sixfold greater than rHisExoS. Relative to rHisExoS, ΔN222 had a similar affinity for NAD, a threefold greater affinity for SBTI, and a four- to eightfold greater k(cat) 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, ΔN222 did not chromatograph as an aggregate, which showed that the amino-terminal 99 amino acids of exoenzyme S were responsible for the aggregation phenotype.