During
iron starvation the Gram-negative pathogenic bacterium Pseudomonas aeruginosa makes the nonribosomal
peptide siderophore pyochelin by a four
protein, 11 domain assembly line, involving a cascade of acyl-S-
enzyme intermediates on the PchE and PchF subunits that are elongated, heterocyclized, reduced, and N-methylated before release. Purified PchG is shown to be an
NADPH-dependent
reductase for the hydroxyphenylbisthiazoline-S-PchF acyl
enzyme, regiospecifically converting one of the dihydroheterocyclic thiazoline rings to a
thiazolidine. The K(m) for the PchG
protein is 1 microM, and the k(cat) for throughput to
pyochelin is 2 min(-1). The
nitrogen of the newly generated
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 PCP domain. A presumed
methyltransferase (MT) domain embedded in the PchF subunit catalyzes this N-methylation. Mutation of a conserved G to R in the MT core motif abolishes MT activity and subsequent chain release from PchF. The thioesterase (TE) domain of PchF catalyzes hydrolytic release of the fully 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 hydroxyphenylbisthiazolinyl-COOH (HPTT-COOH) chain reduction and N-methylation. A mutation in the PchF TE domain does not catalyze autodeacylation and release of the pyochelinyl-S-
enzyme. Thus, full reconstitution of the
nonribosomal peptide synthetase assembly line by purified
protein components has been obtained for production of this tandem bisheterocyclic
siderophore.