Phosphopantetheinylation and Specificity of Acyl Carrier Proteins in the Mupirocin Biosynthetic Cluster

Abstract

Acyl carrier proteins are vital for the biosynthesis of fatty acids and polyketides. The mupirocin biosynthetic cluster of Pseudomonas fluorescens encodes eleven type I ACPs embedded in its multifunctional polyketide synthase (PKS) proteins plus five predicted type II ACPs (mAcpA-E) that are known to be essential for mupirocin biosynthesis by deletion and complementation analysis. MupN is a putative Sfp-type phosphopantetheinyl transferase. Overexpression of three type I and three type II mupirocin ACPs in Escherichia coli, with or without mupN, followed by mass spectroscopy revealed that MupN can modify both mupirocin type I and type II ACPs to their holo-form. The endogenous phosphopantetheinyl transferase of E. coli modified mAcpA but not mAcpC or D. Overexpression of the type II ACPs in macp deletion mutants of the mupirocin producer P. fluorescens 10586 showed that they cannot substitute for each other while hybrids between mAcpA and mAcpB indicated that, at least for mAcpB, the C-terminal domain determines functional specificity. Amino acid alignments identified mACPs A and D as having C-terminal extensions. Mutation of these regions generated defective ACPs, the activity of which could be restored by overexpression of the macp genes on separate plasmids.

Defining the genes for the final steps in biosynthesis of the complex polyketide antibiotic mupirocin by Pseudomonas fluorescens NCIMB10586

“…Our previous studies with mupN , which encodes an Sfp-like PPTase 24 , had shown only that it is needed to activate at least some of the ACPs encoded in the mup cluster 18,25 . Shields et al .…”

The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life

“…162 Indeed, when mupN was deleted, mupirocin production was abolished, and it was shown in vitro that both type I and type II ACPs were modified by this PPTase. 43 In contrast to P. aeruginosa , P. fluorescens has two PPTases, PfPcpS and MupN, at its disposal, and it remains a question whether PaPcpS and PfPcpS have different substrate specificity.…”

Polyketide β-branching: diversity, mechanism and selectivity

“…10). 28,[60][61][62] The initially reported incorporation of a cleaved acetate group was conrmed through a combination of isotope labelling and gene cluster comparison. 25,63 The mechanism of such b-branching was elucidated in the bacillaene biosynthetic pathway in vitro by Calderone et al using a combination of biochemical assays and mass spectrometry.…”