FUNCTIONAL DOMAINS OF PSEUDOMONAS-AERUGINOSA EXOENZYME-S

Recombinant exoenzyme S (rHisExoS) of Pseudomonas aeruginosa was expre ssed in Escherichia coli as a soluble, cytosolic His fusion protein. r HisExoS was purified by Ni2+-affinity chromatography in the presence o f protease inhibitors without detectable degradation. rHisExoS possess ed a specific activity (within twofold) for the factor-activating exoe nzyme S-dependent ADP-ribosylation of soybean trypsin inhibitor (SBTI) similar to that of native exoenzyme S. Analysis of several deletion p eptides showed that Delta N222, which encoded the carboxyl-terminal 22 2 amino acids of exoenzyme S, possessed factor-activating exoenzyme S- dependent ADP-ribosyltransferase activity. Delta N222 catalyzed the AD P-ribosylation of SBTI at a rate sixfold greater than rHisExoS. Relati ve to rHisExoS, Delta N222 had a similar affinity for NAD, a threefold greater affinity for SBTI, and a four- to eightfold greater k(cat) fo r the ADP-ribosylation of SBTI. Like native exoenzyme S, rHisExoS chro matographed as an aggregate with an apparent molecular mass of >300 kD a. In contrast, Delta N222 did not chromatograph as an aggregate, whic h showed that the amino-terminal 99 amino acids of exoenzyme S were re sponsible for the aggregation phenotype.