RESUMO
L1 is a dizinc subclass B3 metallo-ß-lactamase (MBL) that hydrolyzes most ß-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed ß-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the ß-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1ß-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1ß-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1ß-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3' leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (ß) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and ß-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.
Assuntos
Antibacterianos , Inibidores de beta-Lactamases , beta-Lactamas , Humanos , Antibacterianos/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , beta-Lactamas/química , beta-Lactamas/metabolismo , beta-Lactamas/farmacologia , Carbapenêmicos/metabolismo , Cristalografia , Cinética , Stenotrophomonas maltophilia/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológicoRESUMO
Variants of isocitrate dehydrogenase (IDH) 1 and 2 (IDH1/2) alter metabolism in cancer cells by catalyzing the NADPH-dependent reduction of 2-oxoglutarate (2OG) to (2R)-hydroxyglutarate. However, it is unclear how derivatives of 2OG can affect cancer cell metabolism. Here, we used synthetic C3- and C4-alkylated 2OG derivatives to investigate the substrate selectivities of the most common cancer-associated IDH1 variant (R132H IDH1), of two cancer-associated IDH2 variants (R172K IDH2, R140Q IDH2), and of WT IDH1/2. Absorbance-based, NMR, and electrochemical assays were employed to monitor WT IDH1/2 and IDH1/2 variant-catalyzed 2OG derivative turnover in the presence and absence of 2OG. Our results reveal that 2OG derivatives can serve as substrates of the investigated IDH1/2 variants, but not of WT IDH1/2, and have the potential to act as 2OG-competitive inhibitors. Kinetic parameters reveal that some 2OG derivatives, including the natural product 3-methyl-2OG, are equally or even more efficient IDH1/2 variant substrates than 2OG. Furthermore, NMR and mass spectrometry studies confirmed IDH1/2 variant-catalyzed production of alcohols in the cases of the 3-methyl-, 3-butyl-, and 3-benzyl-substituted 2OG derivatives; a crystal structure of 3-butyl-2OG with an IDH1 variant (R132C/S280F IDH1) reveals active site binding. The combined results highlight the potential for (i) IDH1/2 variant-catalyzed reduction of 2-oxoacids other than 2OG in cells, (ii) modulation of IDH1/2 variant activity by 2-oxoacid natural products, including some present in common foods, (iii) inhibition of IDH1/2 variants via active site binding rather than the established allosteric mode of inhibition, and (iv) possible use of IDH1/2 variants as biocatalysts.
Assuntos
Isocitrato Desidrogenase , Ácidos Cetoglutáricos , Humanos , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/farmacologia , Neoplasias/metabolismo , Especificidade por Substrato , Ligação Proteica/efeitos dos fármacos , CristalografiaRESUMO
Small-ring cage hydrocarbons are popular bioisosteres (molecular replacements) for commonly found para-substituted benzene rings in drug design1. The utility of these cage structures derives from their superior pharmacokinetic properties compared with their parent aromatics, including improved solubility and reduced susceptibility to metabolism2,3. A prime example is the bicyclo[1.1.1]pentane motif, which is mainly synthesized by ring-opening of the interbridgehead bond of the strained hydrocarbon [1.1.1]propellane with radicals or anions4. By contrast, scaffolds mimicking meta-substituted arenes are lacking because of the challenge of synthesizing saturated isosteres that accurately reproduce substituent vectors5. Here we show that bicyclo[3.1.1]heptanes (BCHeps), which are hydrocarbons for which the bridgehead substituents map precisely onto the geometry of meta-substituted benzenes, can be conveniently accessed from [3.1.1]propellane. We found that [3.1.1]propellane can be synthesized on a multigram scale, and readily undergoes a range of radical-based transformations to generate medicinally relevant carbon- and heteroatom-substituted BCHeps, including pharmaceutical analogues. Comparison of the absorption, distribution, metabolism and excretion (ADME) properties of these analogues reveals enhanced metabolic stability relative to their parent arene-containing drugs, validating the potential of this meta-arene analogue as an sp3-rich motif in drug design. Collectively, our results show that BCHeps can be prepared on useful scales using a variety of methods, offering a new surrogate for meta-substituted benzene rings for implementation in drug discovery programmes.
Assuntos
Compostos Bicíclicos com Pontes , Desenho de Fármacos , Heptanos , Ânions/química , Benzeno/química , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/química , Descoberta de Drogas , Heptanos/síntese química , Heptanos/química , Pentanos/síntese química , Pentanos/química , SolubilidadeRESUMO
Ivosidenib, an inhibitor of isocitrate dehydrogenase 1 (IDH1) R132C and R132H variants, is approved for the treatment of acute myeloid leukaemia (AML). Resistance to ivosidenib due to a second site mutation of IDH1 R132C, leading to IDH1 R132C/S280F, has emerged. We describe biochemical, crystallographic, and cellular studies on the IDH1 R132C/S280F and R132H/S280F variants that inform on the mechanism of second-site resistance, which involves both modulation of inhibitor binding at the IDH1 dimer-interface and alteration of kinetic properties, which enable more efficient 2-HG production relative to IDH1 R132C and IDH1 R132H. Importantly, the biochemical and cellular results demonstrate that it should be possible to overcome S280F mediated resistance in AML patients by using alternative inhibitors, including some presently in phase 2 clinical trials.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Isocitrato Desidrogenase , Leucemia Mieloide Aguda , Resistencia a Medicamentos Antineoplásicos/genética , Glicina/análogos & derivados , Glicina/uso terapêutico , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Mutação , Piridinas/uso terapêuticoRESUMO
Isopenicillin N synthase (IPNS) catalyzes formation of the ß-lactam and thiazolidine rings of isopenicillin N from its linear tripeptide l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) substrate in an iron- and dioxygen (O2)-dependent four-electron oxidation without precedent in current synthetic chemistry. Recent X-ray free-electron laser studies including time-resolved serial femtosecond crystallography show that binding of O2 to the IPNS-Fe(II)-ACV complex induces unexpected conformational changes in α-helices on the surface of IPNS, in particular in α3 and α10. However, how substrate binding leads to conformational changes away from the active site is unknown. Here, using detailed 19F NMR and electron paramagnetic resonance experiments with labeled IPNS variants, we investigated motions in α3 and α10 induced by binding of ferrous iron, ACV, and the O2 analog nitric oxide, using the less mobile α6 for comparison. 19F NMR studies were carried out on singly and doubly labeled α3, α6, and α10 variants at different temperatures. In addition, double electron-electron resonance electron paramagnetic resonance analysis was carried out on doubly spin-labeled variants. The combined spectroscopic and crystallographic results reveal that substantial conformational changes in regions of IPNS including α3 and α10 are induced by binding of ACV and nitric oxide. Since IPNS is a member of the structural superfamily of 2-oxoglutarate-dependent oxygenases and related enzymes, related conformational changes may be of general importance in nonheme oxygenase catalysis.
Assuntos
Oxirredutases , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Compostos Ferrosos/química , Ferro/química , Óxido Nítrico/química , Oxirredutases/química , Oxirredutases/genética , Oxigênio/química , Oxigenases/metabolismo , Penicilinas/biossíntese , Penicilinas/química , Conformação Proteica , Especificidade por Substrato , Tiazolidinas/químicaRESUMO
ß-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine ß-lactamase (SBL)-catalyzed hydrolysis. The scope of ß-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfonederived acylenzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acylenzyme complexes.
Assuntos
Compostos Azabicíclicos , Inibidores de beta-Lactamases , Penicilinas , Sulfonas , Triazóis , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/químicaRESUMO
Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.
Assuntos
Elétrons , Oxirredutases , Catálise , Domínio Catalítico , Cristalografia por Raios X , Compostos Férricos , Humanos , Lasers , Oxirredutases/química , Oxigênio/química , Penicilinas/química , Penicilinas/metabolismo , Especificidade por SubstratoRESUMO
Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.
Assuntos
Cristalografia por Raios X/métodos , Enzimas/química , Enzimas/metabolismo , Animais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Galinhas , Cristalografia por Raios X/instrumentação , Desenho de Equipamento , Modelos Moleculares , Muramidase/química , Muramidase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
Macrotermitine termites have domesticated fungi in the genus Termitomyces as their primary food source using predigested plant biomass. To access the full nutritional value of lignin-enriched plant biomass, the termite-fungus symbiosis requires the depolymerization of this complex phenolic polymer. While most previous work suggests that lignocellulose degradation is accomplished predominantly by the fungal cultivar, our current understanding of the underlying biomolecular mechanisms remains rudimentary. Here, we provide conclusive omics and activity-based evidence that Termitomyces employs not only a broad array of carbohydrate-active enzymes (CAZymes) but also a restricted set of oxidizing enzymes (manganese peroxidase, dye decolorization peroxidase, an unspecific peroxygenase, laccases, and aryl-alcohol oxidases) and Fenton chemistry for biomass degradation. We propose for the first time that Termitomyces induces hydroquinone-mediated Fenton chemistry (Fe2+ + H2O2 + H+ â Fe3+ + â¢OH + H2O) using a herein newly described 2-methoxy-1,4-dihydroxybenzene (2-MH2Q, compound 19)-based electron shuttle system to complement the enzymatic degradation pathways. This study provides a comprehensive depiction of how efficient biomass degradation by means of this ancient insect's agricultural symbiosis is accomplished. IMPORTANCE Fungus-growing termites have optimized the decomposition of recalcitrant plant biomass to access valuable nutrients by engaging in a tripartite symbiosis with complementary contributions from a fungal mutualist and a codiversified gut microbiome. This complex symbiotic interplay makes them one of the most successful and important decomposers for carbon cycling in Old World ecosystems. To date, most research has focused on the enzymatic contributions of microbial partners to carbohydrate decomposition. Here, we provide genomic, transcriptomic, and enzymatic evidence that Termitomyces also employs redox mechanisms, including diverse ligninolytic enzymes and a Fenton chemistry-based hydroquinone-catalyzed lignin degradation mechanism, to break down lignin-rich plant material. Insights into these efficient decomposition mechanisms reveal new sources of efficient ligninolytic agents applicable for energy generation from renewable sources.
Assuntos
Biomassa , Isópteros/microbiologia , Lignina/metabolismo , Estresse Oxidativo , Termitomyces/enzimologia , Termitomyces/metabolismo , Animais , Ecossistema , Microbioma Gastrointestinal , Perfilação da Expressão Gênica , Genoma Fúngico , Oxirredução , Plantas/metabolismo , Plantas/microbiologia , Simbiose , Termitomyces/classificação , Termitomyces/genéticaRESUMO
Metallo-ß-lactamases (MBLs) can efficiently catalyze the hydrolysis of all classes of ß-lactam antibiotics except monobactams. While serine-ß-lactamase (SBL) inhibitors (e.g., clavulanic acid, avibactam) are established for clinical use, no such MBL inhibitors are available. We report on the synthesis and mechanism of inhibition of N-sulfamoylpyrrole-2-carboxylates (NSPCs) which are potent inhibitors of clinically relevant B1 subclass MBLs, including NDM-1. Crystallography reveals that the N-sulfamoyl NH2 group displaces the dizinc bridging hydroxide/water of the B1 MBLs. Comparison of crystal structures of an NSPC and taniborbactam (VRNX-5133), presently in Phase III clinical trials, shows similar binding modes for the NSPC and the cyclic boronate ring systems. The presence of an NSPC restores meropenem efficacy in clinically derived E. coli and K. pneumoniae blaNDM-1. The results support the potential of NSPCs and related compounds as efficient MBL inhibitors, though further optimization is required for their clinical development.
Assuntos
Escherichia coli , beta-Lactamases , Antibacterianos/farmacologia , Ácidos Borínicos , Ácidos Carboxílicos , Inibidores de beta-Lactamases/farmacologiaRESUMO
Penems have demonstrated potential as antibacterials and ß-lactamase inhibitors; however, their clinical use has been limited, especially in comparison with the structurally related carbapenems. Faropenem is an orally active antibiotic with a C-2 tetrahydrofuran (THF) ring, which is resistant to hydrolysis by some ß-lactamases. We report studies on the reactions of faropenem with carbapenem-hydrolysing ß-lactamases, focusing on the class A serine ß-lactamase KPC-2 and the metallo ß-lactamases (MBLs) VIM-2 (a subclass B1 MBL) and L1 (a B3 MBL). Kinetic studies show that faropenem is a substrate for all three ß-lactamases, though it is less efficiently hydrolysed by KPC-2. Crystallographic analyses on faropenem-derived complexes reveal opening of the ß-lactam ring with formation of an imine with KPC-2, VIM-2, and L1. In the cases of the KPC-2 and VIM-2 structures, the THF ring is opened to give an alkene, but with L1 the THF ring remains intact. Solution state studies, employing NMR, were performed on L1, KPC-2, VIM-2, VIM-1, NDM-1, OXA-23, OXA-10, and OXA-48. The solution results reveal, in all cases, formation of imine products in which the THF ring is opened; formation of a THF ring-closed imine product was only observed with VIM-1 and VIM-2. An enamine product with a closed THF ring was also observed in all cases, at varying levels. Combined with previous reports, the results exemplify the potential for different outcomes in the reactions of penems with MBLs and SBLs and imply further structure-activity relationship studies are worthwhile to optimise the interactions of penems with ß-lactamases. They also exemplify how crystal structures of ß-lactamase substrate/inhibitor complexes do not always reflect reaction outcomes in solution.
Assuntos
Antibacterianos/química , Inibidores de beta-Lactamases/química , beta-Lactamases/química , beta-Lactamas/química , Antibacterianos/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Hidrólise , Klebsiella pneumoniae/enzimologia , Ligação Proteica , Pseudomonas aeruginosa/enzimologia , Stenotrophomonas maltophilia/enzimologia , Inibidores de beta-Lactamases/metabolismo , beta-Lactamases/metabolismo , beta-Lactamas/metabolismoRESUMO
Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitro-gen cryo-stream at 100â K) enable, is data collection of di-oxy-gen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O2 diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for di-oxy-gen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the 'sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5â min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent l-arginine hy-droxy-lase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.
RESUMO
Terpenoids are enormously diverse, but our knowledge of their biosynthesis and functions is limited. Here we report on a terpene synthase (DdTPS8)-cytochrome P450 (CYP521A1) gene cluster that produces a novel C12 trisnorsesquiterpene and affects the development of Dictyostelium discoideum. DdTPS8 catalyzes the formation of a sesquiterpene discoidol, which is undetectable from the volatile bouquet of wild type D. discoideum. Interestingly, a DdTPS8 knockout mutant lacks not only discoidol, but also a putative trisnorsesquiterpene. This compound was hypothesized to be derived from discoidol via cytochrome P450 (CYP)-catalyzed oxidative cleavage. CYP521A1, which is clustered with DdTPS8, was identified as a top candidate. Biochemical assays demonstrated that CYP521A1 catalyzes the conversion of discoidol to a novel trisnorsesquiterpene named discodiene. The DdTPS8 knockout mutant exhibited slow progression in development. This study points to the untapped diversity of natural products made by D. discoideum, which may have diverse roles in its development and chemical ecology.
Assuntos
Alquil e Aril Transferases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Dictyostelium/enzimologia , Dictyostelium/crescimento & desenvolvimento , Sesquiterpenos/metabolismo , Alquil e Aril Transferases/genética , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/genética , Dictyostelium/genética , Dictyostelium/metabolismo , Família MultigênicaRESUMO
The therapeutic properties of complex terpenes often depend on the stereochemistry of their functional groups. However, stereospecific chemical synthesis of terpenes is challenging. To overcome this challenge, metabolic engineering can be employed using enzymes with suitable stereospecific catalytic activity. Here we used a combinatorial metabolic engineering approach to explore the stereospecific modification activity of the Artemisia annua artemisinic aldehyde ∆11(13) double bond reductase2 (AaDBR2) on products of the feverfew sesquiterpene biosynthesis pathway (GAS, GAO, COS and PTS). This allowed us to produce dihydrocostunolide and dihydroparthenolide. For dihydroparthenolide we demonstrate that the preferred order of biosynthesis of dihydroparthenolide is by reduction of the exocyclic methylene of parthenolide, rather than through C4-C5 epoxidation of dihydrocostunolide. Moreover, we demonstrate a promiscuous activity of feverfew CYP71CB1 on dihydrocostunolide and dihydroparthenolide for the production of 3ß-hydroxy-dihydrocostunolide and 3ß-hydroxy-dihydroparthenolide, respectively. Combined, these results offer new opportunities for engineering novel sesquiterpene lactones with potentially improved medicinal value.
Assuntos
Artemisia annua , Engenharia Metabólica , Oxirredutases , Proteínas de Plantas , Sesquiterpenos/metabolismo , Tanacetum parthenium , Artemisia annua/enzimologia , Artemisia annua/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tanacetum parthenium/enzimologia , Tanacetum parthenium/genéticaRESUMO
Covering: up to 2017 2-Oxoglutarate (2OG) dependent oxygenases and the homologous oxidase isopenicillin N synthase (IPNS) play crucial roles in the biosynthesis of ß-lactam ring containing natural products. IPNS catalyses formation of the bicyclic penicillin nucleus from a tripeptide. 2OG oxygenases catalyse reactions that diversify the chemistry of ß-lactams formed by both IPNS and non-oxidative enzymes. Reactions catalysed by the 2OG oxygenases of ß-lactam biosynthesis not only involve their typical hydroxylation reactions, but also desaturation, epimerisation, rearrangement, and ring-forming reactions. Some of the enzymes involved in ß-lactam biosynthesis exhibit remarkable substrate and product selectivities. We review the roles of 2OG oxygenases and IPNS in ß-lactam biosynthesis, highlighting opportunities for application of knowledge of their roles, structures, and mechanisms.
Assuntos
Oxigenases de Função Mista/metabolismo , Oxirredutases/metabolismo , beta-Lactamas/metabolismo , Carbapenêmicos/biossíntese , Cefalosporinas/biossíntese , Ácidos Cetoglutáricos/metabolismo , Oxigenases de Função Mista/química , Oxigenases/metabolismo , beta-Lactamas/químicaRESUMO
Two bacterial diterpene synthases from the actinomycete Allokutzneria albata were investigated, resulting in the identification of the structurally unprecedented compound spiroalbatene from the first and cembreneâ A from the second enzyme. Both enzymes were thoroughly investigated in terms of their mechanisms by isotope labeling experiments, site-directed mutagenesis, and variation of the metal cofactors and pHâ value. For spiroalbatene synthase, the pH- and Mn2+ -dependent formation of the side product thunbergol was observed, which is biosynthetically linked to spiroalbatene.
Assuntos
Actinomycetales/enzimologia , Alquil e Aril Transferases/metabolismo , Diterpenos/metabolismo , Alquil e Aril Transferases/química , Diterpenos/química , Concentração de Íons de Hidrogênio , Estrutura MolecularRESUMO
The product obtained in vitro from a diterpene synthase encoded in the genome of the bacterium Chitinophaga pinensis, an enzyme previously reported to have germacrene A synthase activity during heterologous expression in Escherichia coli, was identified by extensive NMR-spectroscopic methods as 18-hydroxydolabella-3,7-diene. The absolute configuration of this diterpene alcohol and the stereochemical course of the terpene synthase reaction were addressed by isotopic labelling experiments. Heterologous expression of the diterpene synthase in Nicotiana benthamiana resulted in the production of 18-hydroxydolabella-3,7-diene also in planta, while the results from the heterologous expression in E. coli were shown to be reproducible, revealing that the expression of one and the same terpene synthase in different heterologous hosts may yield different terpene products.
RESUMO
GATA-type transcription factors (TFs) such as the nitrogen regulators AreA and AreB, or the light-responsive TFs WC-1 and WC-2, play global roles in fungal growth and development. The conserved GATA TF NsdD is known as an activator of sexual development and key repressor of conidiation in Aspergillus nidulans, and as light-regulated repressor of macroconidia formation in Botrytis cinerea. In the present study, we functionally characterized the NsdD ortholog in Fusarium fujikuroi, named Csm1. Deletion of this gene resulted in elevated microconidia formation in the wild-type (WT) and restoration of conidiation in the non-sporulating velvet mutant Δvel1 demonstrating that Csm1 also plays a role as repressor of conidiation in F. fujikuroi. Furthermore, biosynthesis of the PKS-derived red pigments, bikaverin and fusarubins, is de-regulated under otherwise repressing conditions. Cross-species complementation of the Δcsm1 mutant with the B. cinerea ortholog LTF1 led to full restoration of WT-like growth, conidiation and pigment formation. In contrast, the F. fujikuroi CSM1 rescued only the defects in growth, the tolerance to H2O2 and virulence, but did not restore the light-dependent differentiation when expressed in the B. cinerea Δltf1 mutant. Microarray analysis comparing the expression profiles of the F. fujikuroi WT and the Δcsm1 mutant under different nitrogen conditions revealed a strong impact of this GATA TF on 19 of the 47 gene clusters in the genome of F. fujikuroi. One of the up-regulated silent gene clusters is the one containing the sesquiterpene cyclase-encoding key gene STC1. Heterologous expression of STC1 in Escherichia coli enabled us to identify the product as the volatile bioactive compound (-)-germacrene D.
RESUMO
Two diterpene cyclases, one from the social amoeba Dictyostelium discoideum and the other from the bacterium Streptomyces clavuligerus, with products containing a Z-configured double bond between the original C2 and C3 of geranylgeranyl diphosphate, were extensively investigated for their mechanisms through isotopic labelling experiments. The participation of geranyllinalyl diphosphate, in analogy to the role of linalyl and nerolidyl diphosphate for mono- and sesquiterpene biosynthesis, as an intermediate towards diterpenes with a Z-configured C2=C3 double bond is discussed.