In vitro reconstitution of the Pseudomonas aeruginosa nonribosomal peptidesynthesis of pyochelin: Characterization of backbone tailoring thiazoline reductase and N-methyltransferase activities

During iron starvation the Gram-negative pathogenic bacterium Pseudomonas a eruginosa makes the nonribosomal peptide siderophore pyochelin by a four pr otein, 11 domain assembly line, involving a cascade of acyl-S-enzyme interm ediates on the PchE and PchF subunits that are elongated, heterocyclized, r educed, and N-methylated before release. Purified PchG is shown to be an NA DPH-dependent reductase for the hydroxyphenylbisthiazoline-S-PchF acyl enzy me, regiospecifically converting one of the dihydroheterocyclic thiazoline rings to a thiazolidine. The K-m for the PchG protein is 1 muM, and the k(c at) for throughput to pyochelin is 2 min(-1). The nitrogen of the newly gen erated thiazolidine ring can be N-methylated upon addition of SAM, to yield the mature pyochelin chain still tethered as a pyochelinyl-S-PchF at the P CP domain. A presumed methyltransferase (MT) domain embedded in the PchF su bunit catalyzes this N-methylation. Mutation of a conserved G to R in the M T core motif abolishes MT activity and subsequent chain release from PchF. The thioesterase (TE) domain of PchF catalyzes hydrolytic release of the fu lly mature pyochelinyl chain to produce the pyochelin siderophore at a rate of 2 min(-1), at least 30-40-fold faster than in the absence of hydroxyphe nylbisthiazolinyl-COOH (HPTT-COOH) chain reduction and N-methylation. A mut ation in the PchF TE domain does not catalyze autodeacylation and release o f the pyochelinyl-S-enzyme. Thus, full reconstitution of the nonribosomal p eptide synthetase assembly line by purified protein components has been obt ained for production of this tandem bisheterocyclic siderophore.