Bacterial aromatic polyketides that include many antibiotic and antitumor therapeutics are biosynthesized by the type II polyketide synthase (PKS), which consists of 5 – 10 stand-alone enzymatic domains. Hedamycin, an antitumor antibiotic polyketide, is uniquely primed with a hexadienyl group generated by a type I PKS followed by coupling to a downstream type II PKS to biosynthesize a 24-carbon polyketide, whose C9 position is reduced by hedamycin type II ketoreductase (hedKR). HedKR is homologous to the actinorhodin KR (actKR), for which we have conducted extensive structural studies previously. How hedKR can accommodate a longer polyketide substrate than the actKR, and the molecular basis of its regio- and stereospecificities, is not well understood. Here we present a detailed study of hedKR that sheds light on its specificity. Sequence alignment of KRs predicts that hedKR is less active than actKR, with significant differences in substrate/inhibitor recognition. In vitro and in vivo assays of hedKR confirmed this hypothesis. The hedKR crystal structure further provides the molecular basis for the observed differences between hedKR and actKR in the recognition of substrates and inhibitors. Instead of the 94-PGG-96 motif observed in actKR, hedKR has the 92-NGG-94 motif, leading to S-dominant stereospecificity, whose molecular basis can be explained by the crystal structure. Together with mutations, assay results, docking simulations, and the hedKR crystal structure, a model for the observed regio- and stereospecificities is presented herein that elucidates how different type II KRs recognize substrates with different chain lengths, yet precisely reduce only the C9-carbonyl group. The molecular features of hedKR important for regio- and stereospecificities can potentially be applied to biosynthesize new polyketides via protein engineering that rationally controls polyketide ketoreduction.
The pentadecaketide fredericamycin has the longest carbon chain backbone among polycyclic aromatic polyketide antibiotics whose biosynthetic genes have been sequenced. This backbone is synthesized by the bimodular fdm polyketide synthase (PKS). The initiation module is thought to synthesize a C6 intermediate that is then transferred onto the elongation PKS module, which extends it into a C30 poly-β-ketoacyl product. Here we demonstrate that the bimodular fdm PKS as well as its elongation module alone synthesize undecaketides and dodecaketides. Thus, unlike other homologues, the fdm ketosynthase – chain length factor (KS-CLF) heterodimer does not exclusively control the backbone length of its natural product. Using sequence- and structure-based approaches, 48 multiple mutants of the CLF were engineered and analyzed. Unexpectedly, the I134F mutant was unable to turn over, but could initiate and at least partially elongate the polyketide chain. This unprecedented mutant suggests that the KS-CLF heterodimer harbors an as yet uncharacterized chain termination mechanism. Together, our findings reveal fundamental mechanistic differences between the fdm PKS and its well-studied homologues.
Analysis of a genetic module repurposed between yeast and vertebrates reveals that a common antifungal medication is also a potent vascular disrupting agent.
Studies in diverse organisms have revealed a surprising depth to the evolutionary conservation of genetic modules. For example, a systematic analysis of such conserved modules has recently shown that genes in yeast that maintain cell walls have been repurposed in vertebrates to regulate vein and artery growth. We reasoned that by analyzing this particular module, we might identify small molecules targeting the yeast pathway that also act as angiogenesis inhibitors suitable for chemotherapy. This insight led to the finding that thiabendazole, an orally available antifungal drug in clinical use for 40 years, also potently inhibits angiogenesis in animal models and in human cells. Moreover, in vivo time-lapse imaging revealed that thiabendazole reversibly disassembles newly established blood vessels, marking it as vascular disrupting agent (VDA) and thus as a potential complementary therapeutic for use in combination with current anti-angiogenic therapies. Importantly, we also show that thiabendazole slows tumor growth and decreases vascular density in preclinical fibrosarcoma xenografts. Thus, an exploration of the evolutionary repurposing of gene networks has led directly to the identification of a potential new therapeutic application for an inexpensive drug that is already approved for clinical use in humans.
Yeast cells and vertebrate blood vessels would not seem to have much in common. However, we have discovered that during the course of evolution, a group of proteins whose function in yeast is to maintain cell walls has found an alternative use in vertebrates regulating angiogenesis. This remarkable repurposing of the proteins during evolution led us to hypothesize that, despite the different functions of the proteins in humans compared to yeast, drugs that modulated the yeast pathway might also modulate angiogenesis in humans and in animal models. One compound seemed a particularly promising candidate for this sort of approach: thiabendazole (TBZ), which has been in clinical use as a systemic antifungal and deworming treatment for 40 years. Gratifyingly, our study shows that TBZ is indeed able to act as a vascular disrupting agent and an angiogenesis inhibitor. Notably, TBZ also slowed tumor growth and decreased vascular density in human tumors grafted into mice. TBZ’s historical safety data and low cost make it an outstanding candidate for translation to clinical use as a complement to current anti-angiogenic strategies for the treatment of cancer. Our work demonstrates how model organisms from distant branches of the evolutionary tree can be exploited to arrive at a promising new drug.
The 1.51 Å resolution X-ray crystal structure of the trans-acyltransferase (AT) from the “AT-less” disorazole synthase (DSZS), and its acetate complex at 1.35 Å resolution, are reported. Separately, comprehensive alanine scanning mutagenesis of one of its acyl carrier protein substrates (ACP1 from DSZS) led to the identification of a conserved Asp45 residue on the ACP, which contributes to the substrate specificity of this unusual enzyme. Together, these experimental findings were used to derive a model for the selective association of the DSZS AT and its ACP substrate. Towards the goal of structurally characterizing the AT-ACP interface, a strategy was developed for covalently cross-linking active site Ser→Cys mutant of the DSZS AT to its ACP substrate, and for purifying the resulting AT-ACP complex to homogeneity. The S86C DSZS AT mutant was found to be functional, albeit with a 200-fold lower transacylation efficiency than its wild-type counterpart. Our findings provide new insights as well as new opportunities for high-resolution analysis of an important protein-protein interface in polyketide synthases.
Techniques that can effectively separate protein–peptide complexes from free peptides have shown great value in MHC–peptide binding studies. However, most of the available techniques are limited to measuring the binding of a single peptide to MHC molecule. As antigen presentation in vivo involves both endogenous ligands and exogenous antigens, the deconvolution of multiple binding events necessitates the implementation of a more powerful technique. Here we show that capillary electrophoresis coupled to fluorescence detection (CE-FL) can resolve multiple MHC peptide binding events owing to its superior resolution and the ability to simultaneously monitor multiple emission channels. We utilized CE-FL to investigate competition and displacement of endogenous peptides by an immunogenic gluten peptide for binding to HLA-DQ2. Remarkably, this immunogenic peptide could displace CLIP peptides from the DQ2 binding site at neutral but not acidic pH. This unusual ability of the gluten peptide supports a direct loading mechanism of antigen presentation in extracellular environment, a property that could explain the antigenicity of dietary gluten in celiac disease.
protein-peptide interactions; multiple ligands; antigen presentation; electrophoresis; fluorescence
Celiac sprue is an inflammatory disease of the small intestine caused by dietary gluten and treated by adherence to a lifelong gluten-free diet. The recent identification of immunodominant gluten peptides, the discovery of their cogent properties, and the elucidation of the mechanisms by which they engender immunopathology in genetically-susceptible individuals have advanced our understanding of the molecular pathogenesis of this complex disease, enabling the rational design of new therapeutic strategies. The most clinically advanced of these is oral enzyme therapy, in which enzymes capable of proteolyzing gluten (i.e. glutenases) are delivered to the alimentary tract of a celiac sprue patient to detoxify ingested gluten in situ. In this chapter, we discuss the key challenges for discovery and preclinical development of oral enzyme therapies for celiac sprue. Methods for lead identification, assay development, gram-scale production and formulation, and lead optimization for next-generation proteases are described and critically assessed.
Coeliac disease is a widespread, lifelong disorder for which dietary control represents the only accepted form of therapy. There is an unmet need for non-dietary therapies to treat this condition. Most ongoing and emerging drug discovery programmes are based on the understanding that coeliac disease is caused by an inappropriate T-cell-mediated immune response to dietary gluten proteins. Recent genome-wide association studies lend further support to this pathogenic model. The central role of human leukocyte antigen genes has been validated, and a number of new risk loci have been identified, most of which are related to the biology of T cells and antigen-presenting cells. Here we review the status of potential non-dietary therapies under consideration for coeliac disease. We conclude that future development of novel therapies will be aided by the identification of new, preferably non-invasive, surrogate markers for coeliac disease activity.
Coeliac disease; gluten; T cell; HLA; transglutaminase; drug; therapy
The polyketide antibiotic frenolicin B harbors a biosynthetically intriguing benzoisochromanequinone core, and has been shown to exhibit promising antiparasitic activity against Eimeria tenella. To facilitate further exploration of its chemistry and biology, we constructed a biosynthetic route to frenolicin B in the heterologous host Streptomyces coelicolor CH999, despite the absence of key enzymes in the identified frenolicin gene cluster. Together with our understanding of the underlying polyketide biosynthetic pathway, this heterologous production system was exploited to produce analogs modified at the C15 position. Both the natural product and these analogs inhibited the growth of Toxoplasma gondii in a manner that reveals sensitivity to the length of the C15 substituent. The ability to construct a functional biosynthetic pathway, despite a lack of genetic information, illustrates the feasibility of a modular approach to engineering medicinally relevant polyketide products.
Inhibitors of human transglutaminase 2 (TG2) are anticipated to be useful in the therapy of a variety of diseases including celiac sprue as well as certain CNS disorders and cancers. A class of 3-acylidene-2-oxoindoles was identified as potent reversible inhibitors of human TG2. Structure-activity relationship analysis of a lead compound led to the generation of several potent, competitive inhibitors. Analogues with significant non-competitive character were also identified, suggesting that the compounds bind at one or more allosteric regulatory sites on this multidomain enzyme. The most active compounds had Ki values below 1.0 µM in two different kinetic assays for human TG2, and may therefore be suitable for investigations into the role of TG2 in physiology and disease in animals.
transglutaminase 2; oxoindole; celiac sprue; structure-activity relationships; allostery
Macrolide antibacterial agents inhibit parasite proliferation by targeting the apicoplast ribosome. Motivated by the long-term goal of identifying antiparasitic macrolides that lack antibacterial activity, we have systematically analyzed the structure-activity relationships among erythromycin analogues and have also investigated the mechanism of action of selected compounds. Two lead compounds, N-benzyl-azithromycin (11) and N-phenylpropyl-azithromycin (30), were identified with significantly higher antiparasitic activity and lower antibacterial activity than erythromycin or azithromycin. Molecular modeling based on the co-crystal structure of azithromycin bound to the bacterial ribosome suggested that a substituent at the N-9 position of desmethyl-azithromycin could improve selectivity due to species-specific interactions with the ribosomal L22 protein. Like other macrolides, these lead compounds display a strong “delayed death phenotype”; however, their early effects on T. gondii replication are more pronounced.
Transglutaminase 2 (TG2) is an allosterically regulated enzyme with transamidating, deamidating and cell signaling activities. It is thought to catalyze sequence-specific deamidation of dietary gluten peptides in the small intestines of celiac disease patients. Because this modification has profound consequences for disease pathogenesis, there is considerable interest in the design of small molecule TG2 inhibitors. Although many classes of TG2 inhibitors have been reported, thus far an animal model for screening them to identify promising celiac drug candidates has remained elusive. Using intraperitoneal administration of the toll-like receptor 3 (TLR3) ligand, polyinosinic-polycytidylic acid (poly(I∶C)), we induced rapid TG2 activation in the mouse small intestine. Dose dependence was observed in the activation of TG2 as well as the associated villous atrophy, gross clinical response, and rise in serum concentration of the IL-15/IL-15R complex. TG2 activity was most pronounced in the upper small intestine. No evidence of TG2 activation was observed in the lung mucosa, nor were TLR7/8 ligands able to elicit an analogous response. Introduction of ERW1041E, a small molecule TG2 inhibitor, in this mouse model resulted in TG2 inhibition in the small intestine. TG2 inhibition had no effect on villous atrophy, suggesting that activation of this enzyme is a consequence, rather than a cause, of poly(I∶C) induced enteropathy. Consistent with this finding, administration of poly(I∶C) to TG2 knockout mice also induced villous atrophy. Our findings pave the way for pharmacological evaluation of small molecule TG2 inhibitors as drug candidates for celiac disease.
We report the synthesis and preliminary characterization of “clickable” inhibitors of human transglutaminase 2 (TG2). These inhibitors possess the 3-halo-4,5-dihydroisoxazole warhead along with bio-orthogonal groups such as azide or alkyne moieties that enable subsequent covalent modification with fluorophores. Their mechanism for inhibition of TG2 is based on halide displacement, resulting in the formation of a stable imino thioether. Inhibition assays against recombinant human TG2 revealed that some of the clickable inhibitors prepared in this study have comparable specificity as benchmark dihydroisoxazole inhibitors reported earlier. At low micromolar concentrations they completely inhibited transiently activated TG2 in a WI-38 fibroblast scratch assay, and could subsequently be used to visualize the active enzyme in situ. The potential use of these inhibitors to probe the role of TG2 in celiac sprue as well as other diseases is discussed.
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 a ~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 either 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.
A-74528 is a recently discovered natural product of Streptomyces sp. SANK 61196 that inhibits 2′,5′-oligoadenylate phosphodiesterase (2′-PDE), a key regulatory enzyme of the interferon pathway. Inhibition of 2′-PDE by A-74528 reduces viral replication, and therefore shows promise as a new type of antiviral drug. The complete A-74528 gene cluster, comprising of 29 open reading frames, was cloned and sequenced, and shown to possess a type II polyketide synthase (PKS) at its core. Its identity was confirmed by analysis of a mutant generated by targeted disruption of a PKS gene, and by functional expression in a heterologous Streptomyces host. Remarkably, it showed exceptional end-to-end sequence identity to the gene cluster responsible for biosynthesis of fredericamycin A, a structurally unrelated antitumor antibiotic with a distinct mode of action. Whereas the fredericamycin producing strain, Streptomyces griseus, produced undetectable quantities of A-74528, the A-74528 gene cluster was capable of producing both antibiotics. The biosynthetic roles of three genes, including one that represents the only qualitative difference between the two gene clusters, were investigated by targeted gene disruption. The implications for the evolution of antibiotics with different biological activities from the same gene cluster are discussed.
The ability to incorporate atypical primer units through the use of dedicated initiation polyketide synthase (PKS) modules offers opportunities to expand molecular diversity of polyketide natural products. Here we identify the initiation PKS module responsible for hexadienyl priming of the antibiotic fredericamycin, and investigate its biochemical properties. We also exploit this PKS module for the design and in vivo biosynthesis of unusually primed analogs of a representative polyketide product, thereby emphasizing its utility to the metabolic engineer.
Erythromycin and related macrolide antibiotics are widely used polyketide natural products. We have evolved an engineered biosynthetic pathway in Escherichia coli that yields erythromycin analogs from simple synthetic precursors. Multiple rounds of mutagenesis and screening led to the identification of new mutant strains with improved efficiency for precursor directed biosynthesis. Genetic and biochemical analysis suggested that the phenotypically relevant alterations in these mutant strains were localized exclusively to the host-vector system, and not to the polyketide synthase. We also demonstrate the utility of this improved system through engineered biosynthesis of a novel alkynyl erythromycin derivative with comparable antibacterial activity to its natural counterpart. In addition to reinforcing the power of directed evolution for engineering macrolide biosynthesis, our studies have identified a new lead substance for investigating structure-function relationships in the bacterial ribosome.
erythromycin; 6-deoxyerythronolide B synthase; precursor directed biosynthesis; polyketide; directed evolution
Acyltransferase (AT) domains of multimodular polyketide synthases are the primary gatekeepers for stepwise incorporation of building blocks into a growing polyketide chain. Each AT domain has two substrates, an α-carboxylated CoA thioester (e.g. malonyl-CoA or methylmalonyl-CoA) and an acyl carrier protein (ACP). Whereas the acyl-CoA specificity of AT domains has been extensively investigated, little is known about their ACP specificity. Guided by recent high-resolution structural insights, we have systematically probed the protein-protein interactions between AT domains, ACP domains and the linkers that flank AT domains. Representative AT domains of the 6-deoxyerythronolide B synthase (DEBS) have greater than 10-fold specificity for their cognate ACP substrates as compared to other ACP domains from the same synthase. Both the flanking (N- and C-terminal) linkers of an AT domain contributed to the efficiency and specificity of transacylation. As a frame of reference, the activity and specificity of a stand-alone AT domain from the “AT-less” disorazole synthase (DSZS) were also quantified. The activity (kcat/KM) of this AT was >250-fold higher than the corresponding values for DEBS AT domains. Although the AT from DSZS discriminated modestly against ACP domains from DEBS, it exhibited >40-fold higher activity in trans in the presence of these heterologous substrates than their natural AT domains. Our results highlight the opportunity for regioselective modification of a polyketide backbone by in trans complementation of inactivated AT domains. They also reinforce the need for more careful consideration of protein-protein interactions in the engineering of these assembly line enzymes.
Hedamycin is an antitumor polyketide antibiotic with unusual biosynthetic features. Earlier sequence analysis of the hedamycin biosynthetic gene cluster implied a role for type I as well as type II polyketide synthases (PKSs). We demonstrate that the hedamycin minimal PKS can synthesize a dodecaketide backbone. The ketosynthase (KS) subunit of this PKS has specificity for both type I and type II acyl carrier proteins (ACPs) with which it collaborates during chain initiation and chain elongation, respectively. The KS receives a C6 primer unit from the terminal ACP domain of HedU (a type I PKS protein) directly, and subsequently interacts with the ACP domain of HedE (a type II PKS protein) during the process of chain elongation. HedE is a bifunctional protein with both ACP and aromatase activity. Its aromatase domain can modulate the chain length specificity of the minimal PKS. Chain length can also be influenced by HedA, the C-9 ketoreductase. Whereas co-expression of the hedamycin minimal PKS and a chain initiation module from the R1128 PKS yields an isobutyryl primed decaketide, the orthologous PKS subunits from the hedamycin gene cluster itself are unable to prime the minimal PKS with a non-acetyl starter unit. Our findings provide new insights into the mechanism of chain initiation and elongation by type II PKSs.
Natural products, produced chiefly by microorganisms and plants, can be large and structurally complex molecules. These molecules are manufactured by cellular assembly lines, in which enzymes construct the molecules in a stepwise fashion. The means by which enzymes interact and work together in a modular fashion to create diverse structural features has been an active area of research; the work has provided insight into the fine details of biosynthesis.
A number of polycyclic aromatic natural products—including several noteworthy anticancer, antibacterial, antifungal, antiviral, antiparasitic, and other medicinally significant substances—are synthesized by polyketide synthases (PKSs) in soil-borne bacteria called actinomycetes. Concerted biosynthetic, enzymological, and structural biological investigations into these modular enzyme systems have yielded interesting mechanistic insights. A core module called the minimal PKS is responsible for synthesizing a highly reactive, protein-bound poly-β-ketothioester chain. In the absence of other enzymes, the minimal PKS also catalyzes chain initiation and release, yielding an assortment of polycyclic aromatic compounds. In the presence of an initiation PKS module, polyketide backbones bearing additional alkyl, alkenyl, or aryl primer units are synthesized, whereas a range of auxiliary PKS enzymes and tailoring enzymes convert the product of the minimal PKS into the final natural product. In this Account, we summarize the knowledge that has been gained regarding this family of PKSs through recent investigations into the biosynthetic pathways of two natural products, actinorhodin and R1128 (A–D).
We also discuss the practical relevance of these fundamental insights for the engineered biosynthesis of new polycyclic aromatic compounds. With a deeper understanding of the biosynthetic process in hand, we can assert control at various stages of molecular construction and thus introduce unnatural functional groups in the process. The metabolic engineer affords a number of new avenues for creating novel molecular structures that will likely have properties akin to their fully natural cousins.
Based on clinical, histopathological and serological similarities to human celiac disease (CD), we recently established the rhesus macaque model of gluten sensitivity. In this study, we further characterized this condition based on presence of anti-tissue transglutaminase 2 (TG2) antibodies, increased intestinal permeability and transepithelial transport of a proteolytically resistant, immunotoxic, 33-residue peptide from α2-gliadin in the distal duodenum of gluten-sensitive macaques.
Six rhesus macaques were selected for study from a pool of 500, including two healthy controls and four gluten-sensitive animals with elevated anti-gliadin or anti-TG2 antibodies as well as history of non-infectious chronic diarrhea. Pediatric endoscope-guided pinch biopsies were collected from each animal's distal duodenum following administration of a gluten-containing diet (GD) and again after remission by gluten-free diet (GFD). Control biopsies always showed normal villous architecture, whereas gluten-sensitive animals on GD exhibited histopathology ranging from mild lymphocytic infiltration to villous atrophy, typical of human CD. Immunofluorescent microscopic analysis of biopsies revealed IgG+ and IgA+ plasma-like cells producing antibodies that colocalized with TG2 in gluten-sensitive macaques only. Following instillation in vivo, the Cy-3-labeled 33-residue gluten peptide colocalized with the brush border protein villin in all animals. In a substantially enteropathic macaque with “leaky” duodenum, the peptide penetrated beneath the epithelium into the lamina propria.
The rhesus macaque model of gluten sensitivity not only resembles the histopathology of CD but it also may provide a model for studying intestinal permeability in states of epithelial integrity and disrepair.
Modularity is a highly sought after feature in engineering design. A modular catalyst is a multi-component system whose parts can be predictably interchanged for functional flexibility and variety. Nearly two decades after the discovery of the first modular polyketide synthase (PKS), we critically assess PKS modularity in the face of a growing body of atomic structural and in vitro biochemical investigations. Both the architectural modularity and the functional modularity of this family of enzymatic assembly lines are reviewed, and the fundamental challenges that lie ahead for the rational exploitation of their full biosynthetic potential are discussed.
Due to their unique ability to cleave immunotoxic gluten peptides endoproteolytically, prolyl endopeptidases (PEPs) are attractive oral therapeutic candidates for protecting celiac sprue patients from the toxic effects of dietary gluten. Enhancing the activity and stability of PEPs under gastric conditions (low pH, high pepsin concentration) is a challenge for protein engineers. Using a combination of sequence- and structure-based approaches together with machine learning algorithms, we have identified improved variants of the Sphingomonas capsulata PEP, a target of clinical relevance. Through two rounds of iterative mutagenesis and analysis, variants with as much as 20% enhanced specific activity at pH 4.5 and 200-fold greater resistance to pepsin were identified. Our results vividly reinforce the concept that conservative changes in proteins, especially in hydrophobic residues within tightly packed regions, can profoundly influence protein structure and function in ways that are difficult to predict entirely from first principles and must therefore be optimized through iterative design and analytical cycles. Incubation with whole wheat bread under simulated gastric conditions also suggests that some variants have pharmacologically significant improvements in gluten detoxification activity.
celiac sprue; prolyl endopeptidase
Tylactone synthase (TYLS) is a modular polyketide synthase that catalyzes the formation of tylactone (1), the parent aglycone precursor of the macrolide antibiotic tylosin. TYLS modules 1 and 2 are responsible for the generation of anti-diketide and triketide intermediates, respectively, each bound to an acyl carrier protein (ACP) domain. Each module harbors a ketoreductase (KR) domain. The stereospecificity of TYLS KR1 and TYLS KR2 has been determined by incubating each of the recombinant ketoreductase domains with reconstituted ketosynthase—acyltransferase [KS][AT] and ACP domains from the 6-deoxyerythronolide B synthase (DEBS) in the presence of the N-acetylcysteamine thioester of syn-(2S,3R)-2-methyl-3-hydroxypentanoate (6), methylmalonyl-CoA, and NADPH resulting in the exclusive formation of the ACP-bound (2R,3R,4S,5R)-2,4-methyl-3,5-dihydroxyhepanoyl triketide, as established by GC-MS analysis of the TMS ether of the derived triketide lactone 7. Both TYLS KR1 and KR2 therefore catalyze the stereospecific reduction of the 2-methyl-3-ketoacyl-ACP substrate from the re-face, with specificity for the reduction of the (2R)-methyl (D) diastereomer. The dehydration that is catalyzed by the dehydratase (DH) domains of TYLS module 2 to give the unsaturated (2E,4S,5R)-2,4-dimethyl-5-hydroxyhept-2-enoyl-ACP2 is therefore a syn elimination of water.
Mesenchymal stromal cells (MSC) markers are expressed on brain tumor-initiating cells involved in the development of hypoxic glioblastoma. Given that MSC can survive hypoxia and that the glucose-6-phosphate transporter (G6PT) provides metabolic control that contributes to MSC mobilization and survival, we investigated the effects of low oxygen (1.2% O2) exposure on G6PT gene expression. We found that MSC significantly expressed G6PT and the glucose-6-phosphatase catalytic subunit β (G6PC-3), while expression of the glucose-6-phosphatase catalytic subunit α (G6PC) and the islet-specific glucose-6-phosphatase catalytic subunit-related protein (G6PC-2) gene expression was low to undetectable. Analysis of the G6PT promoter sequence revealed potential binding sites for the hypoxia inducible factor-1 (HIF-1) α and for the aryl hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator (ARNT) AhR:ARNT. In agreement with this, hypoxia and the hypoxia mimetic cobalt chloride induced the expression of G6PT, vascular endothelial growth factor (VEGF) and HIF-1α. Gene silencing of HIF-1α prevented G6PT and VEGF induction in hypoxic MSC while generation of cells stably expressing HIF-1α resulted in increased endogenous G6PT gene expression. A semi-synthetic analog of the polyketide mumbaistatin, a potent G6PT inhibitor, specifically reduced MSC-HIF-1αcell survival. Collectively, our data suggest that G6PT may account for the metabolic flexibility that enables MSC to survive under conditions characterized by hypoxia and could be specifically targeted within developing tumors.
Mesenchymal stromal cells; hypoxia inducible factor-1 alpha; glucose-6-phosphate transporter; stem cell migration; brain tumor