Purification, priming, and catalytic acylation of carrier protein domains in the polyketide synthase and nonribosomal peptidyl synthetase modules of the HMWP1 subunit of yersiniabactin synthetase

The 207-kDa polyketide synthase (PKS) module (residues 1-1895) and the 143- kDa nonribosomal peptidyl synthetase (NRPS) module (1896-3163) of the 350-k Da HMWP1 subunit of yersiniabactin synthetase have been expressed in and pu rified from Escherichia coil in soluble forms to characterize the acyl carr ier protein (ACP) domain of the PKS module and the homologous peptidyl carr ier protein (PCP3) domain of the NRPS module. The apo-ACP and PCP domains c ould be selectively posttranslationally primed by the E. coli ACPS and EntD phosphopantetheinyl transferases (PPTases), respectively, whereas the Baci llus subtilis PPTase Sfp primed both carrier protein domains in vitro or du ring in vivo coexpression. The holo-NRPS module but not the holo-PK5 module was then selectively aminoacylated with cysteine by the adenylation domain embedded in the HMWP2 subunit of yersiniabactin synthetase, acting in tran s. When the acyltransferase (AT) domain of HMWP1 was analyzed for its abili ty to malonylate the hole carrier protein domains, in cis acylation was fir st detected. Then, in trans malonylation of the excised holo-ACP or holo-PC P3-TE fragments by HMWP1 showed both were malonylated with a 3:1 catalytic efficiency ratio, showing a promiscuity to the AT domain.