[Show abstract][Hide abstract] ABSTRACT: Polyketide synthase (PKS) β-processing domains are responsible for much of the stereochemical complexity of polyketide natural products. Although the importance of β-processing domains has been well noted and significantly explored, key stereochemical details pertaining to cryptic stereochemistry and the impact of remote stereogenic centers have yet to be fully discerned. To uncover the inner workings of ketoreductases (KR) and dehydratases (DH) from the tylosin pathway a didomain composed of TylDH3-KR3 was recombinantly expressed and interrogated with full-length tetraketide substrates to probe the impact of vicinal and distal stereochemistry. In vitro product isolation analysis revealed the products of the cryptic KR as D-alcohols and of the DH as trans-olefins. Steady-state kinetic analysis of the dehydration reaction demonstrated a strict stereochemical tolerance at the β-position as D-configured substrates were processed more than 100 times more efficiently than L-alcohols. Unexpectedly, the kcat/KM values were diminished 14- to 45-fold upon inversion of remote ε- and ζ-stereocenters. This stereochemical discrimination predicted to be driven by a combination of allylic A1,3 strain that likely disfavors binding of the ε-epimer and a loss of electrostatic interactions with the ζ-epimer. Our results strongly suggest that dehydratases may play a role in refining the stereochemical outcomes of preceding modules through their substrate stereospecificity, honing the configurational purity of the final PKS product.
Chemical Science 06/2015; 6(8). DOI:10.1039/C5SC01505G · 9.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metabolic engineering of polyketide synthase (PKS) pathways represents a promising approach to natural products discovery. The dehydratase (DH) domains of PKSs, which generate an α,β-unsaturated bond through a dehydration reaction, have been poorly studied compared to other domains, likely due to the simple nature of the chemical reaction they catalyze and lack of a convenient assay to measure substrate turnover. Herein we report the first steady-state kinetic analysis of a PKS DH domain employing LC-MS/MS analysis for product quantitation. PikDH2 was selected as a model DH domain and its substrate specificity and mechanism was interrogated with a systematic series of synthetic triketide substrates employing nonhydrolyzable thioether linkages as well as by site-directed mutagenesis, evaluation of the pH dependence of catalytic efficiency (Vmax/KM), and through kinetic characterization of a mechanism-based inhibitor. These studies revealed PikDH2 converts D-alcohol substrates to provide trans-olefin products. PikDH2 possesses remarkably strict substrate specificity and is unable to turnover substrates epimeric at the β, γ or δ-positions. The reaction is reversible with equilibrium constants ranging from 1.2-2. Moreover, the enzyme activity is robust and PikDH2 was used on a preparative scale for the chemoenzymatic synthesis of unsaturated triketide products. We also demonstrated PikDH2 has a key ionizable group with a pKa of 7.0 and can be irreversibly inactivated through covalent modification by a mechanism-based inhibitor, which provides a foundation for future structural studies to elucidate substrate-protein interactions.
Journal of the American Chemical Society 06/2015; 137(22). DOI:10.1021/jacs.5b02325 · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Covering: 2000 to 2014Since their first report in 2000, tubulysins have sparked great interest for development as anti-cancer agents due to their exceptionally potent antiproliferative activity. Progress in the discovery and development of tubulysins, especially tubulysin conjugates, has quickly advanced despite limitations in their availability from Nature. In this Highlight, the key research on the isolation and structure determination, biosynthesis, bioactivity, structure-activity relationships (SAR), synthesis, and conjugates of tubulysins is presented.
[Show abstract][Hide abstract] ABSTRACT: Among natural product families, polyketides have shown the most promise for combinatorial biosynthesis of natural product-like libraries. Though recent research in the area has provided many mechanistic revelations, a basic-level understanding of kinetic and substrate tolerability is still needed before the full potential of combinatorial biosynthesis can be realized. We have developed a novel set of chemical probes for the study of ketoreductase domains of polyketide synthases. This chemical tool-based approach was verified using the ketoreductase of pikromycin module 2 (PikKR2) as a model system. Triketide substrates mimics 12 and 13 were designed to increase stability (incorporating a non-hydrolyzable thioether linkage) and minimize non-essential functionality (truncating the phosphopantetheinyl arm). PikKR2 reduction product identities as well as steady-state kinetic parameters were revealed by a combination of LC-MS/MS analysis of synthetic standards and a NADPH consumption assay. The D-hydroxyl product is consistent with bioinformatic analysis, and results from a complimentary biochemical and molecular biological approach. When compared to widely employed substrates in previous studies, diketide 63 and trans-decalone, substrates 12 and 13 showed 2-10 fold lowered KM values (2.4 ± 0.8 and 7.8 ± 2.7 mM, respectively), indicating molecular recognition of intermediate-like substrates. Due to an abundance of the nonreducable enol-tautomer, the kcat values were attenuated by as much as 15-336 fold relative to known substrates. This study reveals the high stereospecificity of PikKR2 in the face of gross substrate permutation, highlighting the utility of a chemical probe-based approach in the study of polyketide ketoreductases.
ACS Chemical Biology 10/2014; 9(12). DOI:10.1021/cb5006883 · 5.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A general strategy to enzymatically label acyl carrier proteins (ACPs) of polyketide synthases has been developed. Incorporation of a chloromethyl ketone or vinyl ketone moiety into polyketide chain elongation intermediate mimics allows for the synthesis of CoA adducts. These CoA adducts undergo enzymatic reaction with Sfp, a phosphopantetheinyl transferase, to afford labeled CurB carrier proteins.
[Show abstract][Hide abstract] ABSTRACT: A stereoselective total synthesis of the cytotoxic natural products tubulysin U, tubulysin V, and its unnatural epimer epi-tubulysin V, is reported. Simplified analogues containing N,N-dimethyl-D-alanine as a replacement for the N-terminal N-Me-pipecolinic acid residue of the tubulysins are also disclosed. Biological evaluation of these natural products and analogues provided key information with regard to structural and stereochemical requirements for antiproliferative activity and tubulin polymerization inhibition.
[Show abstract][Hide abstract] ABSTRACT: The pikromycin polyketide synthase (PKS) of S. venezuelae, which consists of one loading module and six extension modules, is responsible for the formation of the hexaketide narbonolide, a key intermediate in the biosynthesis of the antibiotic pikromycin. S. venezuelae strains in which PikAI, which houses the loading domain and first two modules of the PKS, is either absent or catalytically inactive, produce no pikromycin product. When these strains are grown in the presence of a synthetically prepared triketide product, activated as the N-acetylcysteamine thioester, pikromycin yields are restored to as much as 11 % of that seen in the wild-type strain. Feeding analogues of the triketide intermediate provides pikromycin analogues bearing different alkyl substituents at C13 and C14. One of these analogues, Delta(15,16)-dehydropikromycin, exhibits improved antimicrobial activity relative to pikromycin.
[Show abstract][Hide abstract] ABSTRACT: A series of tubulysin analogs in which one of the stereogenic centers of tubuphenylalanine was eliminated were synthesized. All compounds were tested for antiproliferative activity towards ovarian cancer cells and for inhibition of tubulin polymerization. The dimethyl analogs were generally more active than the desmethyl analogs, and four analogs have tubulin polymerization IC(50) values similar to combretastatin A4 and the hemiasterlin analog HTI-286.
[Show abstract][Hide abstract] ABSTRACT: An efficient route for the synthesis of the tubulysin family of antimitotic peptides was developed. Simplified tubulysin analogues were synthesized to define the minimum pharmacophore required for cytotoxicity. Simplified tubulysin analogues retain significant cytotoxicity and reveal important preliminary structure-activity relationships.
[Show abstract][Hide abstract] ABSTRACT: Heterologous production of natural products in non-native bacteria can be used to increase yields of certain bioactive compounds; however, producing small molecules inside bacteria has numerous limitations. Two reports of the in vitro reconstruction of entire biosynthetic pathways highlight the advantages and challenges of this approach for pathway engineering.
Nature Chemical Biology 10/2007; 3(9):531-2. DOI:10.1038/nchembio0907-531 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An improved total synthesis of narbonolide and its biotransformation to pikromycin is reported. This total synthesis utilized an intramolecular Nozaki-Hiyama-Kishi coupling that significantly improved macrocyclization yields (90-96%) and allowed for differentiation of the C3- and C5-oxidation states. A pikAI deletion mutant of Streptomyces venezuelae was used to biotransform synthetic narbonolide to pikromycin by glycosylation and oxidation in vivo. This integration of synthetic chemistry and engineered biotransformations holds great promise for the synthesis of novel macrolide analogues of biological interest.
The Journal of Organic Chemistry 01/2007; 71(26):9853-6. DOI:10.1021/jo062047u · 4.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polyketides are a diverse class of natural products having important clinical properties, including antibiotic, immunosuppressive and anticancer activities. They are biosynthesized by polyketide synthases (PKSs), which are modular, multienzyme complexes that sequentially condense simple carboxylic acid derivatives. The final reaction in many PKSs involves thioesterase-catalyzed cyclization of linear chain elongation intermediates. As the substrate in PKSs is presented by a tethered acyl carrier protein, introduction of substrate by diffusion is problematic, and no substrate-bound type I PKS domain structure has been reported so far. We describe the chemical synthesis of polyketide-based affinity labels that covalently modify the active site serine of excised pikromycin thioesterase from Streptomyces venezuelae. Crystal structures reported here of the affinity label-pikromycin thioesterase adducts provide important mechanistic insights. These results suggest that affinity labels can be valuable tools for understanding the mechanisms of individual steps within multifunctional PKSs and for directing rational engineering of PKS domains for combinatorial biosynthesis.
Nature Chemical Biology 11/2006; 2(10):531-6. DOI:10.1038/nchembio822 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polyketides are a class of biologically active microbial and plant-derived metabolites that possess a high degree of structural and functional diversity and include many human therapeutics, among them anti-infective and anti-cancer drugs, growth promoters and anti-parasitic agents. The macrolide antibiotics, characterized by a glycoside-linked macrolactone, constitute an important class of polyketides, including erythromycin and the natural ketolide anti-infective agent pikromycin. Here we describe new mechanistic details of macrolactone ring formation catalyzed by the pikromycin polyketide synthase thioesterase domain from Streptomyces venezuelae. A pentaketide phosphonate mimic of the final pikromycin linear chain-elongation intermediate was synthesized and shown to be an active site affinity label. The crystal structures of the affinity-labeled enzyme and of a 12-membered-ring macrolactone product complex suggest a mechanism for cyclization in which a hydrophilic barrier in the enzyme and structural restraints of the substrate induce a curled conformation to direct macrolactone ring formation.
Nature Chemical Biology 11/2006; 2(10):537-42. DOI:10.1038/nchembio824 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The discovery of the clinical effectiveness of erythromycin and azithromycin in inflammatory airway diseases has inspired the discovery and development of macrolides with selective immunomodulatory activity. Erythromycin degradation continues to be a source of novel macrolides with a variety of selective biological activities. New technologies for drug discovery based in the emerging field of combinatorial biosynthesis provide the medicinal chemist with novel approaches toward the discovery of novel macrolides. Recent efforts to integrate synthetic organic medicinal chemistry with combinatorial biosynthesis have expanded the number of techniques available for macrolide synthesis.
Current opinion in drug discovery & development 09/2006; 8(6):741-7. DOI:10.1002/chin.200637215 · 5.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: [reaction: see text] An improved synthesis of (3S)-3-dihydronarbonolide is reported that constitutes a formal total synthesis of the 14-membered macrolactone antibiotic narbonolide. The key step was an intramolecular Nozaki-Hiyama-Kishi coupling to accomplish macrocyclization in improved yield. The high level of convergence will also allow us to rapidly synthesize narbonolide analogues for the study of enzymes in the pikromycin biosynthetic pathway.
The Journal of Organic Chemistry 10/2005; 70(18):7267-72. DOI:10.1021/jo050924a · 4.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The unique ability of the pikromycin (Pik) polyketide synthase to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes underlying polyketide synthesis, specifically the mechanistic details of chain extension, keto group processing, acyl chain release, and macrocyclization. We have synthesized the natural pentaketide and hexaketide chain elongation intermediates as N-acetyl cysteamine (NAC) thioesters and have used them as substrates for in vitro conversions with engineered PikAIII+TE and in combination with native PikAIII (module 5) and PikAIV (module 6) multifunctional proteins. This investigation demonstrates directly the remarkable ability of these monomodules to catalyze one or two chain extension reactions, keto group processing steps, acyl-ACP release, and cyclization to generate 10-deoxymethynolide and narbonolide. The results reveal the enormous preference of Pik monomodules for their natural polyketide substrates and provide an important comparative analysis with previous studies using unnatural diketide NAC thioester substrates.
Journal of the American Chemical Society 07/2005; 127(23):8441-52. DOI:10.1021/ja042592h · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The polyketide synthase-derived pikromycin thioesterase (Pik TE) is unique in its ability to catalyze the cyclization of 12- and 14-membered macrolactones. In this investigation, the total synthesis of the natural hexaketide chain elongation intermediate as its N-acetyl cysteamine (NAC) thioester has been achieved, and its reaction with Pik TE demonstrated the ability of Pik TE to catalyze its macrolactonization to the natural product 10-deoxymethynolide. A steady-state kinetic analysis of the hexaketide chain intermediate with Pik TE was done. A preliminary substrate specificity study with unnatural hexaketide analogues was accomplished, demonstrating the importance of total synthesis in obtaining access to advanced polyketide intermediates. The results show the sensitivity of Pik TE to minor substrate modifications, and illustrate the potential use of thioesterases as versatile macrolactonization catalysts.
Journal of the American Chemical Society 07/2005; 127(25):8910-1. DOI:10.1021/ja0504340 · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fatty acid amide hydrolase (FAAH) degrades neuromodulating fatty acid amides including anandamide (endogenous cannabinoid agonist) and oleamide (sleep-inducing lipid) at their sites of action and is intimately involved in their regulation. Herein we report the discovery of a potent, selective, and efficacious class of reversible FAAH inhibitors that produce analgesia in animal models validating a new therapeutic target for pain intervention. Key to the useful inhibitor discovery was the routine implementation of a proteomics-wide selectivity screen against the serine hydrolase superfamily ensuring selectivity for FAAH coupled with systematic in vivo examinations of candidate inhibitors.
[Show abstract][Hide abstract] ABSTRACT: In pursuit of an apparent literature anomaly, S- and R-6-methyl-6,7-dihydro-2H-benzo[a]quinolizin-2-one-3-carboxylic acids (12 and 22) were synthesized by an unambiguous route from optically active norephedrines, and their antibacterial potencies were measured. Against Gram-negative microorganisms and DNA gyrase a preference for S-absolute configuration was found whereas R-absolute stereochemistry was more active against Gram-positives. These results are in partial conflict with an earlier report. In an attempt to enhance potency, racemic 10-fluoro-9-piperazinyl (35) and related analogues were synthesized by a novel route. The latter analogues were surprisingly unimproved in potency. The implications of these findings are briefly discussed.