Biosynthetic Origin of the Ether Ring in Platensimycin.
ABSTRACT: Platensimycin (PTM) and platencin (PTN) are highly functionalized bacterial diterpenoid natural products that target bacterial and mammalian fatty acid synthases. PTM and PTN feature varying diterpene-derived ketolides that are linked to the same 3-amino-2,4-dihydroxybenzoic acid moiety. As a result, PTM is a selective inhibitor for FabF/FabB, while PTN is a dual inhibitor of FabF/FabB and FabH. We previously determined that the PTM cassette, consisting of five genes found in the ptm, but not ptn, gene cluster, partitions the biosynthesis of the PTM and PTN diterpene-derived ketolides. We now report investigation of the PTM cassette through the construction of diterpene production systems in E. coli and genetic manipulation in the PTM-PTN dual overproducer Streptomyces platensis SB12029, revealing two genes, ptmT3 and ptmO5, that are responsible for the biosynthetic divergence between the PTM and PTN diterpene-derived ketolides. PtmT3, a type I diterpene synthase, was determined to be a (16R)-ent-kauran-16-ol synthase, the first of its kind found in bacteria. PtmO5, a cytochrome P450 monooxygenase, is proposed to catalyze the formation of the characteristic 11S,16S-ether ring found in PTM. Inactivation of ptmO5 in SB12029 afforded the ?ptmO5 mutant SB12036 that accumulated nine PTM and PTN congeners, seven of which were new, including seven 11-deoxy-16R-hydroxy-PTM congeners. The two fully processed PTM analogues showed antibacterial activities, albeit lower than that of PTM, indicating that the ether ring, or minimally the stereochemistry of the hydroxyl group at C-16, is crucial for the activity of PTM.
Project description:Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases. The regio- and stereospecificity of the ether oxygen atom in PTM, which PTN does not have, strongly contribute to the selectivity and potency of PTM. We previously reported the biosynthetic origin of the 11 S,16 S-ether moiety by characterizing the diterpene synthase PtmT3 as a (16 R)- ent-kauran-16-ol synthase and isolating 11-deoxy-16 R-hydroxylated congeners of PTM from the ? ptmO5 mutant. PtmO5, a cytochrome P450, was proposed to catalyze formation of the ether moiety in PTM. Here we report the in vitro characterization of PtmO5, revealing that PtmO5 stereoselectively hydroxylates the C-11 position of the ent-kaurane scaffold resulting in an 11 S,16 R-diol intermediate. The ether moiety, the oxygen of which originates from the P450-catalyzed hydroxylation at C-11, is formed via cyclization of the diol intermediate. This study provides mechanistic insight into ether formation in natural product biosynthetic pathways.
Project description:Thiocarboxylic acid-containing natural products are rare and their biosynthesis and biological significance remain unknown. Thioplatensimycin (thioPTM) and thioplatencin (thioPTN), thiocarboxylic acid congeners of the antibacterial natural products platensimycin (PTM) and platencin (PTN), were recently discovered. Here we report the biosynthetic origin of the thiocarboxylic acid moiety in thioPTM and thioPTN. We identify a thioacid cassette encoding two proteins, PtmA3 and PtmU4, responsible for carboxylate activation by coenzyme A and sulfur transfer, respectively. ThioPTM and thioPTN bind tightly to β-ketoacyl-ACP synthase II (FabF) and retain strong antibacterial activities. Density functional theory calculations of binding and solvation free energies suggest thioPTM and thioPTN bind to FabF more favorably than PTM and PTN. Additionally, thioacid cassettes are prevalent in the genomes of bacteria, implicating that thiocarboxylic acid-containing natural products are underappreciated. These results suggest that thiocarboxylic acid, as an alternative pharmacophore, and thiocarboxylic acid-containing natural products may be considered for future drug discovery.
Project description:Platensimycin (PTM) and platencin (PTN) are potent inhibitors of bacterial fatty acid synthases and have emerged as promising antibacterial drug leads. We previously characterized the PTM and PTN biosynthetic machineries in the Streptomyces platensis producers. We now identify two mechanisms for PTM and PTN resistance in the S. platensis producers-the ptmP3 or ptnP3 gene within the PTM-PTN or PTN biosynthetic cluster and the fabF gene within the fatty acid synthase locus. PtmP3/PtnP3 and FabF confer PTM and PTN resistance by target replacement and target modification, respectively. PtmP3/PtnP3 also represents an unprecedented mechanism for fatty acid biosynthesis in which FabH and FabF are functionally replaced by a single condensing enzyme. These findings challenge the current paradigm for fatty acid biosynthesis and should be considered in future development of effective therapeutics targeting fatty acid synthase.
Project description:The platensimycin (PTM) and platencin (PTN) class of natural products are promising drug leads that target bacterial and mammalian fatty acid synthases. Natural congeners and synthetic analogues of PTM and PTN have been instrumental in determining their structure-activity relationships, with only a few analogues retaining the potencies of PTM and PTN. Here we describe the identification and isolation of two new sulfur-containing PTM congeners (3 and 5) from the engineered dual PTM-PTN overproducing Streptomyces platensis SB12029. Structure elucidation of platensimycin D1 (5), a sulfur-containing PTM pseudo-dimer, revealed the existence of its presumptive thioacid precursor (3). The unstable thioacid 3 was isolated and confirmed by structural characterization of its permethylated product (6). LC-MS analysis of crude extracts of SB12029 confirmed the presence of the thioacid analogue of PTN (4). The minimum inhibitory concentration (MIC) was determined for 5 revealing retention of the strong antibacterial activity of PTM.
Project description:Platensimycin (PTM) and platencin (PTN) are members of a new class of promising drug leads that target bacterial and mammalian fatty acid synthases. We previously cloned and sequenced the PTM and PTN gene clusters, discovered six additional PTM-PTN dual producing strains, and demonstrated the dramatic overproduction of PTM and PTN by inactivating the pathway-specific regulators ptmR1 or ptnR1 in five different strains. Our ability to utilize these PTM-PTN dual overproducing strains was limited by their lack of genetic amenability. Here we report the construction of Streptomyces platensis SB12029, a genetically amenable, in-frame ?ptmR1 dual PTM-PTN overproducing strain. To highlight the potential of this strain for future PTM and PTN biosynthetic studies, we created the ?ptmR1 ?ptmO4 double mutant S. platensis SB12030. Fourteen PTM and PTN congeners, ten of which were new, were isolated from SB12030, shedding new insights into PTM and PTN biosynthesis. PtmO4, a long-chain acyl-CoA dehydrogenase, is strongly implicated to catalyze ?-oxidation of the diterpenoid intermediates into the PTM and PTN scaffolds. SB12029 sets the stage for future biosynthetic and bioengineering studies of the PTM and PTN family of natural products.
Project description:Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. We have previously cloned and sequenced the PTM-PTN dual biosynthetic gene cluster from Streptomyces platensis MA7327 and the PTN biosynthetic gene cluster from S. platensis MA7339, the latter of which is composed of 31 genes encoding PTN biosynthesis, regulation, and resistance. We have also demonstrated that PTM or PTN production can be significantly improved upon inactivation of the pathway-specific regulator ptmR1 or ptnR1 in S. platensis MA7327 or MA7339, respectively. We now report engineered production of PTN and congeners in a heterologous Streptomyces host. Expression constructs containing the ptn biosynthetic gene cluster were engineered from SuperCos 1 library clones and introduced into five model Streptomyces hosts, and PTN production was achieved in Streptomyces lividans K4-114. Inactivation of ptnR1 was crucial for expression of the ptn biosynthetic gene cluster, thereby PTN production, in S. lividans K4-114. Six PTN congeners, five of which were new, were also isolated from the recombinant strain S. lividans SB12606, revealing new insights into PTN biosynthesis. Production of PTN in a model Streptomyces host provides new opportunities to apply combinatorial biosynthetic strategies to the PTN biosynthetic machinery for structural diversity.
Project description:Bacterial fatty acid synthases are promising antibacterial targets against multidrug-resistant pathogens. Platensimycin (PTM) is a potent FabB/FabF inhibitor, while its poor pharmacokinetics hampers the clinical development. In this study, a focused library of PTM derivatives was prepared through thiolysis of PTM oxirane (1), followed by various C-C cross-coupling reactions in high yields. Antibacterial screening of these compounds in vitro yielded multiple hits with improved anti-Staphylococcus activities over PTM. Among them, compounds A1, A3, A17, and A28 exhibited improved antibacterial activities over PTM against methicillin-resistant Staphylococcus aureus (MRSA) in a mouse peritonitis model. Compound A28 was further shown to be effective against MRSA infection in a mouse wound model, in comparison to mupirocin. Therefore, the facile preparation and screening of these PTM derivatives, together with their potent antibacterial activities in vivo, suggest a promising strategy to improve the antibacterial activity and pharmacokinetic properties of PTM.
Project description:The organization of the fatty acid synthetic genes of Haemophilus influenzae Rd is remarkably similar to that of the paradigm organism, Escherichia coli K-12, except that no homologue of the E. coli fabF gene is present. This finding is unexpected, since fabF is very widely distributed among bacteria and is thought to be the generic 3-ketoacyl-acyl carrier protein (ACP) synthase active on long-chain-length substrates. However, H. influenzae Rd contains a homologue of the E. coli fabB gene, which encodes a 3-ketoacyl-ACP synthase required for unsaturated fatty acid synthesis, and it seemed possible that the H. influenzae FabB homologue might have acquired the functions of FabF. E. coli mutants lacking fabF function are unable to regulate the compositions of membrane phospholipids in response to growth temperature. We report in vivo evidence that the enzyme encoded by the H. influenzae fabB gene has properties essentially identical to those of E. coli FabB and lacks FabF activity. Therefore, H. influenzae grows without FabF function. Moreover, as predicted from studies of the E. coli fabF mutants, H. influenzae is unable to change the fatty acid compositions of its membrane phospholipids with growth temperature. We also demonstrate that the fabB gene of Vibrio cholerae El Tor N16961 does not contain a frameshift mutation as was previously reported.
Project description:Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The ?-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis ?-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known ?-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival.
Project description:Inactivation of ptmB1, ptmB2, ptmT2, or ptmC in Streptomyces platensis SB12029, a platensimycin (PTM) and platencin (PTN) overproducer, revealed that PTM and PTN biosynthesis features two distinct moieties that are individually constructed and convergently coupled to afford PTM and PTN. A focused library of PTM and PTN analogues was generated by mutasynthesis in the ?ptmB1 mutant S. platensis SB12032. Of the 34 aryl variants tested, 18 were incorporated with high titers.