A novel function of yeast fatty acid synthase

177

222

4 -Phosphopantetheine transferases (PPTases) transfer the 4 -phosphopantetheine moiety of coenzyme A onto a conserved serine residue of acyl carrier proteins (ACPs) of fatty acid and polyketide synthases as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases. This posttranslational modification converts ACPs and PCPs from their inactive apo into the active holo form. We have investigated the 4 -phosphopantetheinylation reaction in Bacillus subtilis, an organism containing in total 43 ACPs and PCPs but only two PPTases, the acyl carrier protein synthase AcpS of primary metabolism and Sfp, a PPTase of secondary metabolism associated with the nonribosomal peptide synthetase for the peptide antibiotic surfactin. We identified and cloned ydcB encoding AcpS from B. subtilis, which complemented an Escherichia coli acps disruption mutant. B. subtilis AcpS and its substrate ACP were biochemically characterized. AcpS also modified the D-alanyl carrier protein but failed to recognize PCP and an acyl carrier protein of secondary metabolism discovered in this study, designated AcpK, that was not identified by the Bacillus genome project. On the other hand, Sfp was able to modify in vitro all acyl carrier proteins tested. We thereby extend the reported broad specificity of this enzyme to the homologous ACP. This in vitro cross-interaction between primary and secondary metabolism was confirmed under physiological in vivo conditions by the construction of a ydcB deletion in a B. subtilis sfp ؉ strain. The genes coding for Sfp and its homolog Gsp from Bacillus brevis could also complement the E. coli acps disruption. These results call into question the essential role of AcpS in strains that contain a Sfp-like PPTase and consequently the suitability of AcpS as a microbial target in such strains.4Ј-Phosphopantetheine (Ppant) 1 is an essential prosthetic group of several acyl carrier proteins involved in pathways of primary and secondary metabolism. These include acyl carrier proteins (ACPs) of fatty acid synthases (FASs), ACPs of polyketide synthases (PKSs), and peptidyl carrier proteins (PCPs) and aryl carrier proteins of nonribosomal peptide synthetases (NRPSs) (1, 2). The free thiol moiety of Ppant serves to covalently bind the acyl reaction intermediates as thioesters during the multistep assembly of the monomeric precursors, typically acetyl, malonyl, and aminoacyl groups. Ppant fulfills two demands in these biosynthetic pathways. First, the intermediates remain covalently tethered to the multifunctional enzyme templates in an energy-rich linkage. Second, the flexibility and length of Ppant (about 20 Å) facilitates the transport of the intermediates to the spatially distinct reaction centers. The Ppant moiety is derived from coenzyme A (CoA) and posttranslationally transferred onto an invariant serine side chain. This Mg 2ϩ -dependent conversion of the apoproteins to the holoproteins is catalyzed by the 4Ј-phosphopantetheine transferases (PPTases) (1, 2) (see Fig. 1).Most organisms that employ more t...

Section: ј-Phosphopantetheine (Ppant)mentioning

confidence: 87%

4′-Phosphopantetheine Transfer in Primary and Secondary Metabolism of Bacillus subtilis

Mootz¹,

Finking²,

Marahiel

2001

177

222

“…Although class 2 PPTs are monomers consisting of two domains, structural comparison between class 1 and 2 PPTs has demonstrated that they have comparable domain architecture despite the lack of sequence similarity (30). Class 3 PPTs are distinct in that they constitute a specific enzymatic domain integrated within a larger, multifunctional protein, and the PPT of FAS2 in Saccharomyces cerevisiae is the prototypical example (31).…”

Section: Discussionmentioning

confidence: 99%

Acyl Carrier Protein-specific 4′-Phosphopantetheinyl Transferase Activates 10-Formyltetrahydrofolate Dehydrogenase

Strickland

Hoeferlin

Oleinik

et al. 2010

4-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP posttranslational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO 2 . In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G 1 arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions.Folate derivatives function as coenzymes in reactions involving the transfer of one-carbon units and participate in key metabolic processes, including nucleotide biosynthesis and the regeneration of methionine from homocysteine (1). The intracellular folate pool consists of several major reduced folate forms, and the overall balance between different folates is maintained by numerous enzymes that incorporate one-carbon groups into the pool, convert these groups within the pool, or utilize them in biosynthetic reactions. One of the folate enzymes, 10-formyltetrahydrofolate (10-fTHF) 2 dehydrogenase (FDH, aldehyde dehydrogenase 1L1, EC 1.5.1.6), oxidizes the formyl group of 10-fTHF to yield tetrahydrofolate (THF) and CO 2 . This reaction requires NADP ϩ and proceeds through a complex multistep mechanism (2). FDH is expressed in most tissues and is particularly abundant in the liver and kidney; in the former it is present at the level of about 1% of the total soluble cytosolic protein (3).The FDH substrate, 10-fTHF, is used as a formyl group donor in two steps of de novo purine biosynthesis (4, 5). The excess of 10-fTHF, not required for this pathway, can be utilized by FDH to replenish the THF pool, thus making folate available for other reactions of one-carbon metabolism. It has been proposed that the FDH reaction serves as a regulator of intracellular purine levels, preventing the excessive flux of one-carbon groups into this pathway (6). Several other metabolic functions have also been attributed to FDH, including storage of intracellular folate in the form of THF (7); detoxification of intracellular formic acid by removing it as CO 2 (8); and regulation of o...

“…Sfp has proven to recognize every CP tested so far, including PCPs of NRPSs and ACPs of FASs and PKSs. PPTases of the third group act as integrated domains on their cognate ACP of type I FAS, as is the case in Saccharomyces cerevisiae, for example (11) (Fig. 2).…”

mentioning

confidence: 98%

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

Finking

Solsbacher²,

Konz³

et al. 2002

101

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...