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Yersiniabactin synthetase comprises four proteins, YbtE, HMWP1, HMWP2, and YbtU, encompassing seventeen functional domains, twelve catalytic and five carrier, to select, activate, and incorporate salicylate, three cysteines, and one malonyl moiety into the iron chelator yersiniabactin (Ybt). In the present study, yersiniabactin has been reconstituted in vitro from the 4 protein assembly line by the use of eight biosynthetic precursors. The rate of one turnover, comprising 22 chemical operations performed by the assembly line to release the completed Ybt molecule, was determined at 1.4 min(-1). During the course of Ybt production, the elongating acyl-S-enzyme chain was shown to transfer across a nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) interprotein interface and then a PKS/NRPS intraprotein interface. This study on the Ybt synthetase assembly line represents the first complete in vitro reconstitution of a nonribosomal peptide/polyketide hybrid system.

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The six domain, 229 kDa HMWP2 subunit of the Yersinia pestis yersiniabactin (Ybt) synthetase has been expressed in soluble, full-length form in E. coli as a C-terminal His8 construct at low growth temperatures and with attenuated induction. All six domains of this nonribosomal peptide synthetase subunit, three phosphopantetheinylatable carrier protein domains (ArCP, PCP1, PCP2), one adenylation (A) domain, and two cyclization domains (Cy1, Cy2), have been assayed and are functional. Mutants that convert the phosphopantetheinylatable serine residue to alanine in each of the carrier protein domains accumulate acyl-S-enzyme intermediates upstream of the blocked apo carrier protein site. The ArCP mutant cannot be salicylated by the adenylation protein YbtE; the PCP1 mutant releases salicyl-cysteine from thiolysis of the Sal-S-ArCP intermediate; and the PCP2 mutant releases hydroxyphenyl-thiazolinyl-cysteine from the HPT-S-PCP1 acyl enzyme intermediate, all of which demonstrates processivity and directionality of chain growth. Restoration of the ArCP mutant's function was accomplished with the native ArCP fragment added in trans. The wild-type HMWP2 subunit accumulates hydroxyphenyl-4, 2-bithiazolinyl-S-enzyme at its most downstream PCP2 carrier site, presumably for transfer to the next subunit, HMWP1. The A domain was found to activate and transfer to PCP1 and PCP2 not only the natural L-Cys but also S-2-aminobutyrate, L-beta-chloroalanine, and L-Ser, enabling testing of the substrate specificity of the Cy domain. Probes of Cy domain function include mutagenesis of the Cy1 domain's conserved signature motif DX(4)DX(2)S to show that both D residues but not the S are crucial for both amide bond formation and heterocyclization. Also the Cy1 domain would accept an alternate upstream electrophilic donor substrate (2,3-dihydroxybenzoyl-S-ArCP) but would not process any of the three alternate downstream nucleophilic acceptors in place of Cys-S-PCP1, even for the amide bond-forming step in chain elongation.

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