BIOCHEMICAL RELATIONSHIPS BETWEEN THE 53-KILODALTON (EXO53) AND 49-KILODALTON (EXOS) FORMS OF EXOENZYME-S OF PSEUDOMONAS-AERUGINOSA

Genetic studies have shown that the 53-kDa (Exo53) and 49-kDa (ExoS) f orms of exoenzyme S of Pseudomonas aeruginosa are encoded bg separate genes, termed exoT and exoS, respectively. Although ExoS and Exo53 pos sess 76% primary amino acid homology, Exo53 has been shown to express ADP-ribosyltransferase activity at about 0.2% of the specific activity of ExoS. The mechanism for the lower ADP-ribosyltransferase activity of Exo53 relative to ExoS was analyzed by using a recombinant deletion protein which contained the catalytic domain of Exo53, comprising its 223 carboxyl-terminal residues (termed N223-53), N223-53 was expresse d in Escherichia coli as a stable, soluble fusion protein which was pu rified to >80% homogeneity, Under linear velocity conditions, N223-53 catalyzed the FAS (for factor activating exoenzyme S)-dependent ADP-ri bosylation of soybean trypsin inhibitor (SBTI) at 0.4% and of the Ras protein at 1.0% of the rates of catalysis by N222-49. N222-49 is a pro tein comprising the 222 carboxyl-terminal residues of ExoS, which repr esent its catalytic domain, N223-53 possessed binding affinities for N AD and SBTI similar to those of N222-49 (less than fivefold difference s in K(m)s) but showed a lower velocity rate for the ADP-ribosylation of SBTI. This indicated that the primary defect for ADP-ribosylation b y Exo53 resided within its catalytic capacity. Analysis of hybrid prot eins, composed of reciprocal halves of N223-53 and N222-49, localized the catalytic defect to residues between positions 235 and 349 of N223 -53, E385 was also identified as a potential active site residue of Ex o53.