IDENTIFICATION OF GLUTAMIC-ACID-381 AS A CANDIDATE ACTIVE-SITE RESIDUE OF PSEUDOMONAS-AERUGINOSA EXOENZYME-S

Exoenzyme S of Pseudomonas aeruginosa (ExoS) is a member of the family of bacterial ADP-ribosylating exotoxins (bAREs). Site-directed mutage nesis of glutamic acids within the catalytic domain of ExoS (termed De lta N222) allowed the identification of the preferential inactivation of ADP-ribosyltransferase activity by alanine substitution of E381. Th e specific activity of the E381A mutant was 0.02% of wild-type Delta N 222. Delta N222(E381A) retained the requirement of factor activating e xoenzyme S (FAS) activation for the expression of ADP-ribosyltransfera se activity. In contrast, E387A, E399A, and E414A mutants possessed AD P-ribosyltransferase activity similar to that of wild-type Delta N222. Kinetic evaluation of E381A and two other mutants, E381D and E381S, s howed that their primary defect was a lower k(cat) in the ADP-ribosyla tion of soybean trypsin inhibitor (SBTI). The K-m for NAD and SBTI and activation by FAS varied 2- and 10-fold relative to Delta N222. In ad dition, the E381 mutants possessed identical protease patterns during thrombin and trypsin digestion as Delta N222, which indicated that E38 1 mutants had retained their overall conformation. Together, these dat a identify E381 as contributing to the catalytic activity of exoenzyme S.