Characterization of a New Type of Phosphopantetheinyl Transferase for Fatty Acid and Siderophore Synthesis in Pseudomonas aeruginosa

Abstract

Phosphopantetheinyl-dependent carrier proteins are part of fatty-acid synthases (primary metabolism), polyketide synthases, and non-ribosomal peptide synthetases (secondary metabolism). For these proteins to become functionally active, they need to be primed with the 4-phosphopantetheine moiety of coenzyme A by a dedicated phosphopantetheine transferase (PPTase). Most organisms that employ more than one phosphopantetheinyl-dependent pathway also have more than one PPTase. Typically, one of these PPTases is optimized for the modification of carrier proteins of primary metabolism and rejects those of secondary metabolism (AcpS-type PPTases), whereas the other, Sfp-type PPTase, efficiently modifies carrier proteins involved in secondary metabolism. We present here a new type of PPTase, the carrier protein synthase of Pseudomonas aeruginosa, an organism that harbors merely one PPTase, namely PcpS. Gene deletion experiments clearly show that PcpS is essential for growth of P. aeruginosa, and biochemical data indicate its association with both fatty acid synthesis and siderophore metabolism. At first sight, PcpS is a PPTase of the monomeric Sfp-type and was consequently expected to have catalytic properties typical for this type of enzyme. However, in vitro characterization of PcpS with natural protein partners and non-cognate substrates revealed that its catalytic properties differ significantly from those of Sfp. Thus, the situation in P. aeruginosa is not simply the result of the loss of an AcpS-type PPTase. PcpS exhibits high catalytic efficiency with the carrier protein of fatty acid synthesis and shows a reduced although significant conversion rate of the carrier proteins of non-ribosomal peptide synthetases from their apo to holo form. This association with enzymes of primary and secondary metabolism indicates that PcpS belongs to a new sub-class of PPTases. 4Ј-Phosphopantetheine (Ppant)1 -dependent carrier proteins (CP) are the central entity in fatty-acid synthases (FAS), polyketide synthases (PKS), and non-ribosomal peptide synthetases (NRPSs) (1, 2). The superfamily of CP includes the acidic acyl and aryl carrier proteins and the neutral peptidyl carrier proteins (PCPs). They can be part of a larger polypeptide chain or exist as distinct proteins but always fulfill the same job; during the multistep assembly of the product, the reaction intermediates of the growing acyl or polypeptide chain remain covalently tethered to the Ppant cofactor moiety of these proteins. This moiety is about 20 Å in length and enables the bound intermediates to move between the reaction centers of multifunctional proteins. The thioester linkage that is used to bind the intermediates and final product is energy-rich, which facilitates cleavage after the final step of the assembly. After ribosomal synthesis, however, the carrier protein exists only in the inactive apo form. The Ppant moiety is post-translationally transferred from coenzyme A to a conserved serine residue of the CP in a Mg 2ϩ -dependent reaction by a dedicated phosph...

Cloning, Expression, and Characterization of a Human 4′-Phosphopantetheinyl Transferase with Broad Substrate Specificity

“…Recently, both prokaryotic and eukaryotic organisms have been identified that appear to use a single PPTase for servicing carrier proteins associated with both primary and secondary metabolic pathways. For example, Marahiel's team have reported that Pseudomonas aeruginosa harbors but one PPTase that is responsible for the phosphopantetheinylation of the ACP required for fatty acid biosynthesis and the PCPs required for biosynthesis of the non-ribosomally produced peptide siderophores pyoverdin and pyochelin (29). In common with the situation in humans, the PPTase of P. aeruginosa is of the high molecular mass, monomeric class and, of course, this enzyme too, displays a very broad substrate specificity.…”

Evidence for a novel phosphopantetheinyl transferase domain in the polyketide synthase for enediyne biosynthesis

“…4b). Note that although Sfp has been suggested to exhibit substrate preference towards the CPs from secondary metabolic pathways [22], the higher modification efficiency for EACP in comparison to the three CPs from secondary metabolic pathways probably reflects the variation among different FAS-ACPs. Indeed, very different catalytic efficiency has been observed for Sfp towards PCPs from different species, including tycc3-PCP from B. subtilis (k cat = 96 min À1 , k cat /K m = 22.6 min À1 lM À1 ) and GPCP (GrsA-PCP) from B. brevis (k cat = 10.3 min À1 , k cat / K m = 2.5 min À1 lM À1 ) [23,24].…”

The pobA gene of Burkholderia cenocepacia encodes a Group I Sfp-type phosphopantetheinyltransferase required for biosynthesis of the siderophores ornibactin and pyochelin

“…One possibility is that the division within the Sfp-type PPTases reflects a functional difference. However, although EntD and PcpS both fall within the Group II Sfp-type PPTases, only one of them (PcpS) can substitute for AcpS (Flugel et al, 2000;Finking et al, 2002;Barekzi et al, 2004). Similarly, Sfp and PcpS are members of different PPTase groups, but both can substitute for AcpS ( Fig.…”

“…For example, a Bacillus subtilis acpS mutant supports fatty acid biosynthesis, and Bacillus subtilis Sfp will modify E. coli or Bacillus subtilis apo-ACP in vitro (Quadri et al, 1998b;Mootz et al, 2001). In the case of Pseudomonas aeruginosa, the cross-reactivity of the Sfp-type PPTase is obligatory, as only the Sfp-type PPTase (PcpS) is present (Finking et al, 2002;Barekzi et al, 2004).…”