Dehydratase Domain Research Articles

Nutrient-dependent phosphorylation channels lipid synthesis to regulate PPARα

Peroxisome proliferator-activated receptor (PPAR)α is a nuclear receptor that coordinates liver metabolism during fasting. Fatty acid synthase (FAS) is an enzyme that stores excess calories as fat during feeding, but it also activates hepatic PPARα by promoting synthesis of an endogenous ligand. Here we show that the mechanism underlying this paradoxical relationship involves the differential regulation of FAS in at least two distinct subcellular pools: cytoplasmic and membrane-associated. In mouse liver and cultured hepatoma cells, the ratio of cytoplasmic to membrane FAS-specific activity was increased with fasting, indicating higher cytoplasmic FAS activity under conditions associated with PPARα activation. This effect was due to a nutrient-dependent and compartment-selective covalent modification of FAS. Cytoplasmic FAS was preferentially phosphorylated during feeding or insulin treatment at Thr-1029 and Thr-1033, which flank a dehydratase domain catalytic residue. Mutating these sites to alanines promoted PPARα target gene expression. Rapamycin-induced inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), a mediator of the feeding/insulin signal to induce lipogenesis, reduced FAS phosphorylation, increased cytoplasmic FAS enzyme activity, and increased PPARα target gene expression. Rapamycin-mediated induction of the same gene was abrogated with FAS knockdown. These findings suggest that hepatic FAS channels lipid synthesis through specific subcellular compartments that allow differential gene expression based on nutritional status.

Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase

Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to β-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases.

Sulfonyl 3-Alkynyl Pantetheinamides as Mechanism-Based Cross-Linkers of Acyl Carrier Protein Dehydratase

Acyl carrier proteins (ACPs) play a central role in acetate biosynthetic pathways, serving as tethers for substrates and growing intermediates. Activity and structural studies have highlighted the complexities of this role, and the protein-protein interactions of ACPs have recently come under scrutiny as a regulator of catalysis. As existing methods to interrogate these interactions have fallen short, we have sought to develop new tools to aid their study. Here we describe the design, synthesis, and application of pantetheinamides that can cross-link ACPs with catalytic β-hydroxy-ACP dehydratase (DH) domains by means of a 3-alkynyl sulfone warhead. We demonstrate this process by application to the Escherichia coli fatty acid synthase and apply it to probe protein-protein interactions with noncognate carrier proteins. Finally, we use solution-phase protein NMR spectroscopy to demonstrate that sulfonyl 3-alkynyl pantetheinamide is fully sequestered by the ACP, indicating that the crypto-ACP closely mimics the natural DH substrate. This cross-linking technology offers immediate potential to lock these biosynthetic enzymes in their native binding states by providing access to mechanistically cross-linked enzyme complexes, presenting a solution to ongoing structural challenges.

Structure, Activity, and Substrate Selectivity of the Orf6 Thioesterase from Photobacterium profundum

Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.

Organization and characterization of a biosynthetic gene cluster for bafilomycin from Streptomyces griseus DSM 2608

Streptomyces griseus DSM 2608 produces bafilomycin, an antifungal plecomacrolide antibiotic. We cloned and sequenced an 87.4-kb region, including a polyketide synthase (PKS) region, methoxymalonate genes, flavensomycinate genes, and other putative regulatory genes. The 58.5kb of PKS region consisting 12 PKS modules arranged in five different PKS genes, was assumed to be responsible for the biosynthesis of plecomacrolide backbone including 16-membered macrocyclic lactone. All the modules showed high similarities with typical type I PKS genes. However, the starting module of PKS gene was confirmed to be specific for isobutyrate by sequence comparison of an acyltransferase domain. In downstream of PKS region, the genes for methoxymalonate biosynthesis were located, among which a gene for FkbH-like protein was assumed to play an important role in the production of methoxymalonyl-CoA from glyceryl-CoA. Further the genes encoding flavensomycinyl-ACP biosynthesis for the post-PKS tailoring were also found in the upstream of PKS region. By gene disruption experiments of a dehydratase domain of module 12 and an FkbH-like protein, this gene cluster was confirmed to be involved in the biosynthesis of bafilomycin.

Active Site Comparisons and Catalytic Mechanisms of the Hot Dog Superfamily

In 1996, Leesong et al. published the structure of FabA (PDB ID:1MKA), the dehydratase–isomerase enzyme involved in the bacterial type II fatty acid synthase (FAS) system from E. coli.1 The authors described a central, long α-helix “hot dog” surrounded by a bent β-sheet “bun” and so dubbed this the “hot dog fold” (Figure 1). Since that time, roughly 60 proteins have been crystallized and found to have the hot dog fold.2 The core fold topology can be described as an antiparallel β-sheet ordered 1-3-4-5-2. The long hot dog helix is located between strands β1 and β2 (Figure 2D). Some enzymes contain additional β-strands in their “bun”. Figure 1 The Hot Dog Fold Figure 2 Three Types of Hot Dog Folds This enzyme fold serves as a scaffold to execute a variety of reactions involving fatty acid or polyketide thioesters and is found in enzymes of both fatty acid catabolism and anabolism as well as in enzymes of natural product biosynthetic pathways. Every enzyme in this family containing a single hot dog (SHD) fold dimerizes (Figure 2A and D), and two identical active sites are found at the dimeric interface, with key residues in each active site residing on both monomers. Depending on the enzyme and species, these homodimers (protomers) themselves often associate into dimers of dimers or trimers of dimers. (For an excellent review of the various oligomeric states of hot dog enzymes, see Pidugu et al.2) Some hot dog enzymes have two hot dog motifs — thought to arise from gene duplication — and these double hot dog (DHD) enzymes (or domains) have two SHD subdomains that are fused together. In DHD structures, one of the active sites, which both reside at the pseudodimeric interface, has become inactive (Figure 2B and E). The hot dog helix of the C-terminal domain is often kinked; the one in the N-terminal domain remains linear. The oligomeric states of these pseudodimeric enzymes also vary. There are even instances of triple hot dog (THD) enzymes; fungal FAS dehydratase domains, for example, contain such a fold.3,4 In a THD enzyme, the third hot dog region is a domain insertion between the first and second hot dog motifs, which associate with each other in similar manner to the two motifs of a DHD enzyme, including the bent, C-terminal helix. The added domain is highly distorted, such that the hot dog helix is much shorter. It no longer locates within the β-sheet bun and is instead found alongside the β-sheet (Figure 2C and F). THD enzymes also only have a single active site at the pseudodimeric interface of the “normal” hot dog regions. The significance of only one hot dog helix remaining linear in both DHD and THD folds will be discussed later in this review. The secondary structural arrangements of idealized SHD, DHD, and THD folds are compared in the topology diagrams depicted in Figure 2D, E, and F. Because different enzymes have extra helices and strands in addition to the core hot dog and bun shown in the topology diagrams, for the purposes of comparing hot dog folds, we have formulated the following system: Core β-strands are labeled with an Arabic numeral in the same order as they occur in the sequence. Strands found in the second subdomain of DHD folds are treated as if they reside on an SHD with the further designation of a prime symbol. The strands of the inserted third hot dog motif of the THDs are designated with a double prime. Any extra strands in addition to these may be designated with an “x” followed by an Arabic numeral. The hot dog helices of the first, second, or third motifs are labeled αHD, αHD′, αHD″, respectively; additional helices are named by “x” followed by a capital letter. This review will use this notation throughout. The hot dog superfamily includes over a thousand enzymes or domains and has been divided into at least 17 subfamilies based on primary sequence analysis — although this analysis did not capture the majority of DHD and THD enzymes, including animal and fungal FAS domains.5 Hot dog folds are found or predicted in archaea, bacteria, and eukaryotes. Functionally, however, the majority of the superfamily can be primarily divided into two groups: those enzymes performing either dehydration or hydration of thioester substrates and those performing thioester hydrolysis.

A Polyketide Synthase Gene, ACRTS2, Is Responsible for Biosynthesis of Host-Selective ACR-Toxin in the Rough Lemon Pathotype of Alternaria alternata

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of rough lemon (Citrus jambhiri). The structure of ACR-toxin I (MW = 496) consists of a polyketide with an α-dihydropyrone ring in a 19-carbon polyalcohol. Genes responsible for toxin production were localized to a 1.5-Mb chromosome in the genome of the rough lemon pathotype. Sequence analysis of this chromosome revealed an 8,338-bp open reading frame, ACRTS2, that was present only in the genomes of ACR-toxin-producing isolates. ACRTS2 is predicted to encode a putative polyketide synthase of 2,513 amino acids and belongs to the fungal reducing type I polyketide synthases. Typical polyketide functional domains were identified in the predicted amino acid sequence, including β-ketoacyl synthase, acyl transferase, methyl transferase, dehydratase, β-ketoreductase, and phosphopantetheine attachment site domains. Combined use of homologous recombination-mediated gene disruption and RNA silencing allowed examination of the functional role of multiple paralogs in ACR-toxin production. ACRTS2 was found to be essential for ACR-toxin production and pathogenicity of the rough lemon pathotype of A. alternata.

Expression of a Tetradomain Fragment from a Polyunsaturated Fatty Acid Synthase with Dehydratase Activity

Polyunsaturated fatty acids (PUFAs) are important components of human health and important ingredients in biodiesel preparations. PUFAs from deep‐sea bacteria are synthesized by a modular polyketide synthase, which contains several domains including two dehydratase (DH) domains responsible for the introduction of double bonds. In order to study double bond formation in PUFAs, we have expressed and purified the individual protein fragments from a deep‐sea PUFA synthase. Protein constructs were designed using a bioinformatic tool for the prediction of protein linkers which revealed the presence of two previously uncharacterized pseudo‐domains. The resulting design comprising two putative DH domains in tandem was expressed in E coli, purified by chromatography and assayed against surrogate substrates by UV spectroscopy. Results showed that the designed construct is more active against CoA‐linked substrates than for N‐acetylcysteamine thioester, as revealed by reaction kinetics. Additionally, E coli strains engineered to over‐express DH1‐DH2 can produce 3–5 times more FAs than the wild‐type strain, suggesting that dehydration may be rate‐limiting. We anticipate this result can be implemented to drive the production of FAs in bacteria and be a viable option for the production of biodiesel precursors. This work was funded by Grant CHE0953254 from the NSF and MBRS‐RISE Program (R25GM061838) of the UPR‐MSC.

Modeling holo-ACP:DH and holo-ACP:KR complexes of modular polyketide synthases: a docking and molecular dynamics study

BackgroundModular polyketide synthases are multifunctional megasynthases which biosynthesize a variety of secondary metabolites using various combinations of dehydratase (DH), ketoreductase (KR) and enoyl-reductase (ER) domains. During the catalysis of various reductive steps these domains act on a substrate moiety which is covalently attached to the phosphopantetheine (P-pant) group of the holo-Acyl Carrier Protein (holo-ACP) domain, thus necessitating the formation of holo-ACP:DH and holo-ACP:KR complexes. Even though three dimensional structures are available for DH, KR and ACP domains, no structures are available for DH or KR domains in complex with ACP or substrate moieties. Since Ser of holo-ACP is covalently attached to a large phosphopantetheine group, obtaining complexes involving holo-ACP by standard protein-protein docking has been a difficult task.ResultsWe have modeled the holo-ACP:DH and holo-ACP:KR complexes for identifying specific residues on DH and KR domains which are involved in interaction with ACP, phosphopantetheine and substrate moiety. A novel combination of protein-protein and protein-ligand docking has been used to first model complexes involving apo-ACP and then dock the phosphopantetheine and substrate moieties using covalent connectivity between ACP, phosphopantetheine and substrate moiety as constraints. The holo-ACP:DH and holo-ACP:KR complexes obtained from docking have been further refined by restraint free explicit solvent MD simulations to incorporate effects of ligand and receptor flexibilities. The results from 50 ns MD simulations reveal that substrate enters into a deep tunnel in DH domain while in case of KR domain the substrate binds a shallow surface exposed cavity. Interestingly, in case of DH domain the predicted binding site overlapped with the binding site in the inhibitor bound crystal structure of FabZ, the DH domain from E.Coli FAS. In case of KR domain, the substrate binding site identified by our simulations was in proximity of the known stereo-specificity determining residues.ConclusionsWe have modeled the holo-ACP:DH and holo-ACP:KR complexes and identified the specific residues on DH and KR domains which are involved in interaction with ACP, phosphopantetheine and substrate moiety. Analysis of the conservation profile of binding pocket residues in homologous sequences of DH and KR domains indicated that, these results can also be extrapolated to reductive domains of other modular PKS clusters.

Experimental mapping of soluble protein domains using a hierarchical approach

Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.

New Nystatin-Related Antifungal Polyene Macrolides with Altered Polyol Region Generated via Biosynthetic Engineering of Streptomyces noursei

Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.

Isolation and characterization of a reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.

Stereoselectivity of Isolated Dehydratase Domains of the Borrelidin Polyketide Synthase: Implications for cis Double Bond Formation

Not a matter of preference: The polyketide borrelidin contains both a trans and a cis double bond. The corresponding dehydratases in the polyketide synthase are found to have identical preferences for thioester substrates in vitro and to generate trans double bonds, undermining the idea that cis double bonds arise from dehydration of 3S rather than 3R alcohols.

New non-quinone geldanamycin analogs from genetically engineered Streptomyces hygroscopicus

Heat shock protein 90 (Hsp90) is emerging as an important target in cancer therapeutics and several other diseases.1,2 Hsp90 is an abundant and ubiquitously expressed molecular chaperone that is involved in the maturation of multiple client proteins, many of which are involved in regulating cell signaling, proliferation and survival.3,4 Client proteins of Hsp90 include Her-2, Akt, Src, Abl, c-Met and Raf-1, which are currently being targeted for intervention within oncology drug discovery or in clinical development. Although Hsp90 is highly expressed in most cells, Hsp90 inhibitors display remarkable selectivity for cancer cells as compared with normal cells.5,6 Hsp90 is effectively inhibited by benzoquinone ansamycins such as geldanamycin (GM, 1), herbimycin, macbecin and many of their derivatives, which bind to the ATP-binding site in the N-terminal domain.7,8 However, the development of 1 as a clinical agent has so far been limited by its toxicity, especially liver toxicity.9 However, the promising antitumor properties of 1 have spurred initiatives to develop biologically active derivatives.10,11 17-Allylamino-17demethoxyGM (17-AAG) has reduced toxicity compared with 1 and is the most advanced Hsp90 inhibitor in clinical development (Phase II/III).12 17-AAG and its benzoquinone analogs conjugate with glutathione, leading to cellular depletion. This conjugation with sulfur-containing nucleophiles may contribute to the dose-limiting hepatotoxicity of these quinone-containing compounds.13,14 For these reasons, new non-quinone 1 analogs with improved pharmacological profiles are needed.15,16 Recently, we reported the development of non-quinone 1 analogs by a mutasynthetic approach and directed biosynthetic method.16,17 Of these non-quinone 1 analogs, DHQ3 (3), a 15-hydroxyl-17demethoxy non-quinone analog, was found to inhibit Hsp90 ATPase activity more than 1.16 Moreover, during these studies, novel tricyclic 1 analogs were prepared from a genetically engineered strain (AC15) of Streptomyces hygroscopicus.18 Presently, we describe the fermentation of mutant AC15, and the isolation, structural determination and bioactivity of new non-quinone 1 analogs produced by the mutant: DHQ7 (4) and DHQ8 (5). AC15 was constructed by a combinational mutation with site-directed mutagenesis of the first dehydratase domain of the geldanamycin polyketide synthase (PKS) gene (gelA) and a post-PKS modification gene (gel7) of S. hygroscopicus JCM4427, as previously reported.16 The seed medium and production medium (YEME) consisted of sucrose 103.0 g, yeast extract 3.0 g, peptone (Difco, Sparks, MD, USA) 5.0 g, malt extract (Difco) 3.0 g, glucose 10.0 g and MgCl2 6H2O 1.0 g in 1.0 l of distilled water. Spores that developed during growth on ISP4 medium were harvested and inoculated into 300 ml of YEME in a 1.0 l baffled flask and cultured for 3 days at 28 1C. Equal volumes of the seed culture were inoculated into 25 baffled flasks containing 300 ml of YEME medium. Fermentation was subsequently carried out for 7 days at 28 1C and 160 r.p.m. The resulting culture (B8 l) was extracted twice with an equal volume of ethyl acetate (EtOAc). The extract was filtered through a fritted funnel and the resulting filtrate was evaporated in vacuo to yield the EtOAc extract. This extract was partitioned between EtOAc and water. The EtOAc-soluble material (2.7 g) was subjected to silica gel chromatography using a stepwise gradient elution of mixtures of CH2Cl2 and MeOH. The fraction eluted with CH2Cl2:MeOH of 85:15 was further purified by reverse-phase HPLC using YMC-J’sphere ODS-H80 (10 250 mm, 3 ml min 1; YMC, Kyoto, Japan) with a linear gradient from 30 to 100% acetonitrile (MeCN) containing 0.05% trifluoroacetic acid to yield DHQ7 (4; 31.8 mg) and DHQ8 (5; 17.6 mg). Compound 3 (317 mg) was purified from the fraction eluted with CH2Cl2:MeOH (90:10), subjected to passage over a Sephadex LH-20 (GE Healthcare, Buckinghamshire, UK) column using MeOH as eluant and further purified by HPLC (YMC J’sphere ODS-H80, 10 250 mm, MeOH-H2O (0.05% trifluoroacetic acid) gradient, 3 ml min 1).

Insights into Radicicol Biosynthesis via Heterologous Synthesis of Intermediates and Analogs

Resorcylic acid lactones are fungal polyketides that display diverse biological activities, with the potent Hsp90 inhibitor radicicol being an important representative member. Two fungal iterative polyketide synthases (IPKSs), Rdc5, the highly reducing IPKS, and Rdc1, the nonreducing IPKS, are required for the biosynthesis of radicicol in Pochonia chlamydosporia. In this study, the complete reconstitution of Rdc5 and Rdc1 activities both in vitro and in Saccharomyces cerevisiae uncovered the earliest resorcylic acid lactone intermediate of the radicicol biosynthetic pathway, (R)-monocillin II. The enzymatic synthesis of (R)-monocillin II confirmed the exquisite timing of the Rdc5 enoyl reductase domain. Using precursor-directed biosynthesis, the chemical modularity of the dual IPKS system was determined. Rdc1 readily accepted an N-acetylcysteamine thioester mimic of the reduced pentaketide product of Rdc5 to synthesize (R)-monocillin II with four additional iterations of polyketide elongation, indicating the C2' ketone group found in (R)-monocillin II is incorporated via the functions of Rdc1 instead of Rdc5. The involvement of the thioesterase domain in Rdc1 in macrolactonization was confirmed through both site-directed mutagenesis and domain deletion. The Rdc1 thioesterase domain was also shown to be tolerant of the opposite stereochemistry of the terminal hydroxyl nucleophile, demonstrated in the precursor-directed synthesis of the enantiomeric (S)-monocillin II. Finally, reconstitution of the halogenase Rdc2 was demonstrated both in vivo and in vitro in the synthesis of pochonin D and a new halogenated analog 6-chloro, 7',8'-dehydrozearalenol.

Stereospecificity of the Dehydratase Domain of the Erythromycin Polyketide Synthase

The dehydratase (DH) domain of module 4 of the 6-deoxyerythronolide B synthase (DEBS) has been shown to catalyze an exclusive syn elimination/syn addition of water. Incubation of recombinant DH4 with chemoenzymatically prepared anti-(2R,3R)-2-methyl-3-hydroxypentanoyl-ACP (2a-ACP) gave the dehydration product 3-ACP. Similarly, incubation of DH4 with synthetic 3-ACP resulted in the reverse enzyme-catalyzed hydration reaction, giving an ∼3:1 equilbrium mixture of 2a-ACP and 3-ACP. Incubation of a mixture of propionyl-SNAC (4), methylmalonyl-CoA, and NADPH with the DEBS β-ketoacyl synthase-acyl transferase [KS6][AT6] didomain, DEBS ACP6, and the ketoreductase domain from tylactone synthase module 1 (TYLS KR1) generated in situ anti-2a-ACP that underwent DH4-catalyzed syn dehydration to give 3-ACP. DH4 did not dehydrate syn-(2S,3R)-2b-ACP, syn-(2R,3S)-2c-ACP, or anti-(2S,3S)-2d-ACP generated in situ by DEBS KR1, DEBS KR6, or the rifamycin synthase KR7 (RIFS KR7), respectively. Similarly, incubation of a mixture of (2S,3R)-2-methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester (2b-SNAC), methylmalonyl-CoA, and NADPH with DEBS [KS6][AT6], DEBS ACP6, and TYLS KR1 gave anti-(2R,3R)-6-ACP that underwent syn dehydration catalyzed by DEBS DH4 to give (4R,5R)-(E)-2,4-dimethyl-5-hydroxy-hept-2-enoyl-ACP (7-ACP). The structure and stereochemistry of 7 were established by GC-MS and LC-MS comparison of the derived methyl ester 7-Me to a synthetic sample of 7-Me.

Mechanism and Stereospecificity of a Fully Saturating Polyketide Synthase Module: Nanchangmycin Synthase Module 2 and Its Dehydratase Domain

Recombinant nanchangmycin synthase module 2 (NANS module 2), with the thioesterase domain from the 6-deoxyerythronolide B synthase (DEBS TE) appended to the C-terminus, was cloned and expressed in Escherichia coli. Incubation of NANS module 2+TE with (±)-2-methyl-3-keto-butyryl-N-acetylcysteamine thioester (1), the SNAC analog of the natural ACP-bound substrate, with methylmalonyl-CoA (MM-CoA) in the absence of NADPH gave 3,5,6-trimethyl-4-hydroxypyrone (2), identified by direct comparison with synthetic 2 by radio-TLC-phosphorimaging and LC-ESI(+)-MS-MS. The reaction showed k(cat) 0.5 ± 0.1 min(-1) and K(m)(1) 19 ± 5 mM at 0.5 mM MM-CoA and k(cat)(app) 0.26 ± 0.02 min(-1) and K(m)(MM-CoA) 0.11 ± 0.02 mM at 8 mM 1. Incubation in the presence of NADPH generated the fully saturated triketide chain elongation product as a 5:3 mixture of (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (3a) and the diastereomeric (2S,4S)-3b. The structure and stereochemistry of each product was established by comparison with synthetic 3a and 3b by a combination of radio-TLC-phosphorimaging and LC-ESI(-)-MS-MS, as well as chiral capillary GC-MS analysis of the corresponding methyl esters 3a-Me and 3b-Me. The recombinant dehydratase domain from NANS module 2, NANS DH2, was shown to catalyze the formation of an (E)-double bond by syn-dehydration of the ACP-bound substrate anti-(2R,3R,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl-ACP6 (4), generated in situ by incubation of (2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (5), methylmalonyl-CoA, and NADPH with the recombinant [KS6][AT6] didomain and ACP6 from DEBS module 6 along with the ketoreductase from the tylactone synthase module 1 (TYLS KR1). These results also indirectly establish the stereochemistry of the reactions catalyzed by the KR and enoylreductase (ER) domains of NANS module 2.

Detection of Microcystin-Producing Cyanobacteria in Missisquoi Bay, Quebec, Canada, Using Quantitative PCR

Toxic cyanobacterial blooms, as well as their increasing global occurrence, pose a serious threat to public health, domestic animals, and livestock. In Missisquoi Bay, Lake Champlain, public health advisories have been issued from 2001 to 2009, and local microcystin concentrations found in the lake water regularly exceeded the Canadian drinking water guideline of 1.5 microg liter(-1). A quantitative PCR (Q-PCR) approach was developed for the detection of blooms formed by microcystin-producing cyanobacteria. Primers were designed for the beta-ketoacyl synthase (mcyD(KS)) and the first dehydratase domain (mcyD(DH)) of the mcyD gene, involved in microcystin synthesis. The Q-PCR method was used to track the toxigenic cyanobacteria in Missisquoi Bay during the summers of 2006 and 2007. Two toxic bloom events were detected in 2006: more than 6.5 x 10(4) copies of the mcyD(KS) gene ml(-1) were detected in August, and an average of 4.0 x 10(4) copies ml(-1) were detected in September, when microcystin concentrations were more than 4 microg liter(-1) and approximately 2 microg liter(-1), respectively. Gene copy numbers and total microcystin concentrations (determined by enzyme-linked immunosorbent assay [ELISA]) were highly correlated in the littoral (r = 0.93, P < 0.001) and the pelagic station (r = 0.87, P < 0.001) in 2006. In contrast to the situation in 2006, a cyanobacterial bloom occurred only in late summer-early fall of 2007, reaching only 3 x 10(2) mcyD(KS) copies ml(-1), while the microcystin concentration was barely detectable. The Q-PCR method allowed the detection of microcystin-producing cyanobacteria when toxins and toxigenic cyanobacterial abundance were still below the limit of detection by high-pressure liquid chromatography (HPLC) and microscopy. Toxin gene copy numbers grew exponentially at a steady rate over a period of 7 weeks. Onshore winds selected for cells with a higher cell quota of microcystin. This technique could be an effective approach for the routine monitoring of the most at-risk water bodies.

A mechanism based protein crosslinker for acyl carrier protein dehydratases

Recent advances in the structural study of fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic enzymes have illuminated our understanding of modular enzymes of the acetate pathway. However, one significant and persistent challenge in such analyses is resolution of the acyl carrier protein (ACP), a small (∼9 kDa) protein to which biosynthetic intermediates are tethered throughout the biosynthetic cycle. Here we report a chemoenzymatic crosslinking strategy in which the installation of a historical suicide substrate scaffold upon the 4′-phosphopantetheine (PPant) arm of the ACP is used to capture the active site of acyl carrier protein dehydratase (DH) domains in FAS. Through the synthesis of a small panel of related probes we identify structural features essential for ACP–DH crosslinking, and apply gel-based assays to demonstrate the stability as well as purification strategies for isolation of the chemoenzymatically modified ACP. Applying these carrier protein crosslinking techniques to the structural analysis of FAS and PKS complexes has the potential to provide snapshots of these biosynthetic assembly lines at work.

Insights into Bacterial 6-Methylsalicylic Acid Synthase and Its Engineering to Orsellinic Acid Synthase for Spirotetronate Generation

The enzymes 6-methylsalicylic acid (6-MSA) synthases (6-MSASs) are involved in the building of an aryl moiety in many bioactive secondary metabolites produced by fungi and bacteria. Using the bacterial 6-MSAS ChlB1 in the biosynthesis of spirotetronate antibiotic chlorothricin (CHL) as a model, functional analysis of its dehydratase (DH) and ketoreductase (KR) domains by site-specific mutagenesis revealed that selective ketoreduction is not essential for polyketide chain extension. Promiscuity of the ketoacylsynthase domain in beta functionality recognition allows for engineering ChlB1 to an orsellinic acid (OSA) synthase (OSAS) by inactivating KR at the active site. The engineered ChlB1 is compatible with the enzymes for late-stage tailoring in CHL biosynthesis, featuring specific protein recognitions that facilitate variable aryl group incorporation. The resulting spirotetronates, which bear an OSA-derived aryl group, exhibited antibacterial activities comparable to those of the parent products.