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-kDa HMWP1 subunit of yersiniabactin synthetase have been expressed in and purified from Escherichia coli in soluble forms to characterize the acyl carrier protein (ACP) domain of the PKS module and the homologous peptidyl carrier protein (PCP(3)) domain of the NRPS module. The apo-ACP and PCP domains could be selectively posttranslationally primed by the E. coli ACPS and EntD phosphopantetheinyl transferases (PPTases), respectively, whereas the Bacillus subtilis PPTase Sfp primed both carrier protein domains in vitro or during in vivo coexpression. The holo-NRPS module but not the holo-PKS module was then selectively aminoacylated with cysteine by the adenylation domain embedded in the HMWP2 subunit of yersiniabactin synthetase, acting in trans. When the acyltransferase (AT) domain of HMWP1 was analyzed for its ability to malonylate the holo carrier protein domains, in cis acylation was first detected. Then, in trans malonylation of the excised holo-ACP or holo-PCP(3)-TE fragments by HMWP1 showed both were malonylated with a 3:1 catalytic efficiency ratio, showing a promiscuity to the AT domain.
Suo
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Tseng
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CC
Walsh
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CT
D000213:Acyl Carrier Protein / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000215:Acylation; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D033862:Iron-Binding Proteins; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism; D015394:Molecular Structure; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D033902:Periplasmic Binding Proteins; D010636:Phenols; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011994:Recombinant Proteins; D017262:Siderophores / Q000378:metabolism*; D013844:Thiazoles; D015010:Yersinia pestis / Q000201:enzymology*
D000213:Acyl Carrier Protein; D001059:Apoproteins; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D020035:Holoenzymes; D033862:Iron-Binding Proteins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D033902:Periplasmic Binding Proteins; D010636:Phenols; D021122:Protein Subunits; D011994:Recombinant Proteins; D017262:Siderophores; D013844:Thiazoles; C104398:yersiniabactin; D010204:Pantetheine; D008316:Malonyl Coenzyme A; D000217:Acyltransferases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D003545:Cysteine; C003129:4'-phosphopantetheine