Assembly of the Pseudomonas aeruginosa nonribosomal peptide siderophore pyochelin: In vitro reconstitution of aryl-4,2-bisthiazoline synthetase activity from PchD, PPchE, and PchF

Three Pseudomonas aeruginosa proteins involved in biogenesis of the nonribo somal peptide siderophore pyochelin, PchD, PchE, and PchF, have been expres sed in and purified from Escherichia coli and are found to produce the tric yclic acid hydroxyphenyl-thiazolyl-thiazolinyl-carboxylic acid (HPTT-COOH), an advanced intermediate containing the aryl-4,2-bis-heterocyclic skeleton of the bithiazoline class of siderophores. The three proteins contain thre e adenylation domains, one specific for salicylate activation and two speci fic for cysteine activation, and three carrier protein domains (two in PchE and one in PchF) that undergo posttranslational priming With phosphopantet heine to enable covalent-tethering of salicyl and cysteinyl moieties as acy l-S-enzyme intermediates,Two cyclization domains (Cyl in PchE and Cy2 in Pc hF) create the two amide linkages in the elongating chains and the cyclodeh ydrations of acylcysteine moieties into thiazolinyl rings. The ninth domain , the most downstream domain in PchF, is the chain-terminating, acyl-S-enzy me thioester hydrolase that releases the HPTT-S-enzyme intermediate to the observed tandem bis-heterocyclic acid product. A PchF-thioesterase domain a ctive site double mutant fails to turn over, but a monocyclic hydroxyphenyl -thiazolinyl-cysteine (HPT-Cys) product continues to be released from PchE, allowing assignment of the cascade of acyl-S-enzyme intermediates involved in initiation, elongation, and termination steps.