Expression, purification, and characterization of HMWP2, a 229 kDa, six domain protein subunit of yersiniabactin synthetase

The six domain, 229 kDa HMWP2 subunit of the Yersinia pestis yersiniabactin (Ybt) synthetase has been expressed in soluble, full-length form in E. col i as a C-terminal His8 construct at low growth temperatures and with attenu ated induction. All six domains of this nonribosomal peptide synthetase sub unit, 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 phosphoFante theinylatable serine residue to alanine in each of the carrier protein doma ins accumulate acyl-S-enzyme intermediates upstream of the blocked ape carr ier protein site. The ArCP mutant cannot be salicylated by the adenylation protein YbtE; the PCP1 mutant releases salicyl-cysteine from thiolysis of t he Sal-S-ArCP intermediate; and the PCP2 mutant releases hydroxyphenyl-thia zoIinyl-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 fragme nt added in trans, The wild-type HPI HMWP2 subunit accumulates hydroxypheny l-4.2-bithiazolinyl-S-enzyme at its most downstream PCP2 carrier site, pres umably for transfer to the next subunit, HMWP1. The A domain was found to a ctivate 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 includ e mutagenesis of the Cy1 domain's conserved signature motif DX4-DX2S to sho w that both D residues but not the S are crucial for both amide bond format ion and heterocyclization. Also the Cy1 domain would accept an alternate up stream electrophilic donor substrate (2,3-dihydroxybenzoyl-S-ArCP CP) but w ould not process any of the three alternate downstream nucleophilic accepte rs in place of Cys-S-PCP1, even for the amide bond-forming step in chain el ongation.