malonyl-coenzyme-a and 4--phosphopantetheine

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.

Topics: Acyl Carrier Protein; Acylation; Acyltransferases; Apoproteins; Bacillus subtilis; Bacterial Outer Membrane Proteins; Bacterial Proteins; Cloning, Molecular; Cysteine; Escherichia coli; Holoenzymes; Iron-Binding Proteins; Kinetics; Malonyl Coenzyme A; Molecular Structure; Molecular Weight; Multienzyme Complexes; Pantetheine; Peptide Fragments; Peptide Synthases; Periplasmic Binding Proteins; Phenols; Protein Processing, Post-Translational; Protein Structure, Tertiary; Protein Subunits; Recombinant Proteins; Siderophores; Thiazoles; Yersinia pestis

Two rat liver fatty acid synthetase preparations, containing 1.6 and 2.0 mol of 4'-phosphopantetheine/mol of synthetase, showed specific activity of 2006 and 2140 nmol of NADPH oxidized/min per mg of protein respectively. The two synthetase preparations could be loaded with either 3.3-4.4 mol of [1-14] acetate or 2.9-3.7 mol of [2-14C]malonate, by incubation with either [1-14C] acetyl-CoA or [2-14C]malonyl-CoA. The 4'-phosphopantetheine site could be more than 90% saturated and the serine site about 80% saturated with malonate derived from malonyl-CoA. However, with acetyl-CoA as substrate, binding at both the 4'-phosphopantetheine and cysteine thiol sites did not reach saturation. We interpret these results to indicate that, whereas the equilibrium constant for transfer of substrates between the serine loading site and the 4'-phosphopantetheine site is close to unity, that for transfer of acetyl moieties between the 4'-phosphopantetheine and cysteine sites favours formation of the 4'-phosphopantetheine thioester. Thus, despite the apparent sub-stoichiometric binding of acetate, the results are consistent with a functionally symmetrical model for the fatty acid synthetase which permits simultaneous substrate binding at two separate active centres.

Topics: Acetates; Acetic Acid; Acetyl Coenzyme A; Animals; Binding Sites; Cysteamine; Cysteine; Fatty Acid Synthases; Iodoacetamide; Liver; Malonates; Malonyl Coenzyme A; Pantetheine; Rats