RESUMO
Various nonribosomal peptide synthetases (NRPSs) create structural and functional diversity by incorporating α-hydroxy acids into peptide backbones. Trigonic acid, an unusual cyclopropanol-substituted hydroxy acid, is the source of the molecular warhead of malleicyprol, a critical virulence factor of human and animal pathogens of the Burkholderia pseudomallei (BP) group. The process of selecting and loading this building block remained enigmatic as the NRPS module designated for this task is incomplete. Using a combination of bioinformatics, mutational analyses, targeted metabolomics, and in vitro biochemical assays, we show that two trans-acting enzymes are required to load this central building block onto the modular assembly line. An adenylation-thiolation didomain enzyme (BurJ) activates trigonic acid, followed by the translocation of the enzyme-bound α-hydroxy acid thioester by an FkbH-like protein with a mutated phosphatase domain (BurH). This specialized gateway is the first reported direct loading of an α-hydroxy acid onto a bona fide NRPS module in bacteria and expands the synthetic biology toolbox for the site-specific incorporation of non-canonical building blocks. Moreover, insight into the biochemical basis of virulence factor biosynthesis can provide a foundation for developing enzyme inhibitors as anti-virulence therapeutics against BP pathogen infections.
Assuntos
Hidroxiácidos , Peptídeo Sintases , Peptídeo Sintases/metabolismo , Hidroxiácidos/metabolismo , Hidroxiácidos/química , Burkholderia pseudomallei/enzimologia , Burkholderia pseudomallei/metabolismoRESUMO
Caryoynencin is a toxic and antifungal fatty acid derivative produced by a number of plant-pathogenic and insect-protective bacteria (Trinickia caryophylli and Burkholderia spp.). In addition to the reactive tetrayne unit, the presence of an allylic alcohol moiety is critical for antimicrobial activities. By a combination of mutational analyses, heterologous expression and in vitro reconstitution experiments we show that the cytochromeâ P450 monooxygenase CayG catalyzes the complex transformation of a saturated carbon backbone into an allylic alcohol. Unexpectedly, CayG employs a ferritin-like protein (CayK) or a rubredoxin (CayL) component for electron transport. A desaturation-hydroxylation sequence was deduced from a time-course study and in vitro biotransformations with pathway intermediates, substrate analogues, protegencin congeners from Pseudomonas protegens Pf-5, and synthetic derivatives. This unusual multifunctional oxygenase may inspire future biocatalytic applications.
Assuntos
Sistema Enzimático do Citocromo P-450 , Propanóis , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxilação , OxirreduçãoRESUMO
Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and inâ vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET-domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS-PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli.
Assuntos
Burkholderia/química , Ciclopropanos/metabolismo , Propanóis/metabolismo , Compostos de Sulfônio/metabolismo , Fatores de Virulência/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Burkholderia/enzimologia , Peptídeo Sintases/metabolismo , Policetídeo Sintases/metabolismo , Alinhamento de SequênciaRESUMO
The regioselective functionalization of non-activated carbon atoms such as aliphatic halogenation is a major synthetic challenge. A novel multifunctional enzyme catalyzing the geminal dichlorination of a methyl group was discovered in Aspergillus oryzae (Koji mold), an important fungus that is widely used for Asian food fermentation. A biosynthetic pathway encoded on two different chromosomes yields mono- and dichlorinated polyketides (diaporthin derivatives), including the cytotoxic dichlorodiaporthin as the main product. Bioinformatic analyses and functional genetics revealed an unprecedented hybrid enzyme (AoiQ) with two functional domains, one for halogenation and one for O-methylation. AoiQ was successfully reconstituted inâ vivo and inâ vitro, unequivocally showing that this FADH2 -dependent enzyme is uniquely capable of the stepwise gem-dichlorination of a non-activated carbon atom on a freestanding substrate. Genome mining indicated that related hybrid enzymes are encoded in cryptic gene clusters in numerous ecologically relevant fungi.
Assuntos
Aspergillus oryzae/enzimologia , Fenóis/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Vias Biossintéticas , Fermentação , Genes Fúngicos , Halogenação , Metilação , Fenóis/química , Policetídeos/química , Policetídeos/metabolismo , EstereoisomerismoRESUMO
Bacterial pathogens of the Burkholderia pseudomallei (BP) group cause life-threatening infections in both humans and animals. Critical for the virulence of these often antibiotic-resistant pathogens is the polyketide hybrid metabolite malleicyprol, which features two chains, a short cyclopropanol-substituted chain and a long hydrophobic alkyl chain. The biosynthetic origin of the latter has remained unknown. Here, we report the discovery of novel overlooked malleicyprol congeners with varied chain lengths and identify medium-sized fatty acids as polyketide synthase (PKS) starter units that constitute the hydrophobic carbon tails. Mutational and biochemical analyses show that a designated coenzyme A-independent fatty acyl-adenylate ligase (FAAL, BurM) is essential for recruiting and activating fatty acids in malleicyprol biosynthesis. In vitro reconstitution of the BurM-catalyzed PKS priming reaction and analysis of ACP-bound building blocks reveal a key role of BurM in the toxin assembly. Insights into the function and role of BurM hold promise for the development of enzyme inhibitors as novel antivirulence therapeutics to combat infections with BP pathogens.
Assuntos
Ácidos Graxos , Policetídeo Sintases , Animais , Humanos , Antibacterianos/farmacologia , Bactérias/metabolismo , Policetídeo Sintases/metabolismoRESUMO
Bacteria of the Burkholderia pseudomallei (BP) group pose a global health threat, causing the infectious diseases melioidosis, a common cause of pneumonia and sepsis, and glanders, a contagious zoonosis. A trait of BP bacteria is a conserved gene cluster coding for the biosynthesis of polyketides (malleicyprols) with a reactive cyclopropanol unit that is critical for virulence. Enzymes building this warhead represent ideal targets for antivirulence strategies but the biochemical basis of cyclopropanol formation is unknown. Here we describe the formation of the malleicyprol warhead. We show that BurG, an unusual NAD+-dependent member of the ketol-acid reductoisomerase family, constructs the strained cyclopropanol ring. Biochemical assays and a suite of eight crystal structures of native and mutated BurG with bound analogues and inhibitors provide snapshots of each step of the complex reaction mechanism, involving a concealed oxidoreduction and a C-S bond cleavage. Our findings illustrate a remarkable case of neofunctionalisation, where a biocatalyst from central metabolism has been evolutionarily repurposed for warhead production in pathogens.
Assuntos
Burkholderia mallei , Burkholderia pseudomallei , Mormo , Animais , Bactérias , Burkholderia mallei/genética , Burkholderia pseudomallei/genética , Éteres Cíclicos , Mormo/microbiologia , Mormo/patologia , CavalosRESUMO
In rat exocrine pancreas cells, fluoride treatment causes autophagy resulting from intracisternal granule accumulation. Excessive autophagy might promote a type of programmed cell death different from apoptosis. To clarify how fluoride-induced autophagy and subsequent cell death occurs, we investigated morphological and biochemical changes in exocrine pancreas cells of rats subcutaneously injected with NaF saline solution at 20 mg/kg dose twice daily for 4 days. Intracisternal granule, excessive autophagy and ribosomal degranulation were observed in fluoride-exposed cells, occasionally with necrotic changes. Fluoride-induced rER-stress increased eIF-2alpha phosphorylation and CHOP expression, but did not affect GRP78. Spliced XBP-1 expression was decreased in damaged cells. These findings indicate that rER-stress by intracisternal granule accumulation lead to autophagy in exocrine pancreas cells without UPR, suggesting that signal process of autophagy differs from that of UPR-apoptosis. It is likely that intense degranulation is a turning point that damaged cells change over from autophagy, cell-protective process, to cell-death process.
Assuntos
Apoptose/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Retículo Endoplasmático Rugoso/efeitos dos fármacos , Fluoretos/farmacologia , Pâncreas Exócrino/efeitos dos fármacos , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Retículo Endoplasmático Rugoso/metabolismo , Retículo Endoplasmático Rugoso/ultraestrutura , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Pâncreas Exócrino/enzimologia , Pâncreas Exócrino/ultraestrutura , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Dobramento de Proteína , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fatores de TempoRESUMO
Metabolic profiling and genome mining revealed that anaerobic bacteria have the potential to produce acyloin natural products. In addition to sattazolin A and B, three new sattazolin congeners and a novel acyloin named clostrocyloin were isolated from three strains of Clostridium beijerinckii, a bacterium used for industrial solvent production. Bioactivity profiling showed that the sattazolin derivatives possess antimicrobial activities against mycobacteria and pseudomonads with only low cytotoxicity. Clostrocyloin was found to be mainly active against fungi. The thiamine diphosphate (ThDP)-dependent sattazolin-producing synthase was identified in silico and characterized both in vivo and in in vitro enzyme assays. A related acyloin synthase from the clostrocyloin producer was shown to be responsible for the production of the acyloin core of clostrocyloin. The biotransformation experiments provided first insights into the substrate scope of the clostrocyloin synthase and revealed biosynthetic intermediates.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Álcoois Graxos/química , Álcoois Graxos/farmacologia , Bactérias Anaeróbias/química , Vias Biossintéticas , Clostridium/química , Hexanonas/química , Hexanonas/farmacologia , Humanos , Indóis/química , Indóis/farmacologia , Mycobacterium/efeitos dos fármacos , Infecções por Mycobacterium/tratamento farmacológico , Micoses/tratamento farmacológico , Pseudomonas/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológicoRESUMO
Terpenoid natural products comprise a wide range of molecular architectures that typically result from C-C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Liases/metabolismo , Terpenos/metabolismo , Xenobióticos/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ciclização , Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Hidroxilação , Inativação Metabólica , Índigo Carmim/química , Índigo Carmim/metabolismo , Indóis/química , Indóis/metabolismo , Liases/química , Liases/genética , Estrutura Molecular , Filogenia , Terpenos/química , Xenobióticos/químicaRESUMO
The dimeric structure of kinesin superfamily proteins plays an important role in their motile functions and characteristics. In this study, the coiled-coil-forming property of the stalk region (192-346) of Drosophila ncd, a C-terminal kinesin motor protein, was investigated by synthesizing various peptide fragments. The alpha helicity of a set of 46-residue peptides spanning the stalk region appeared too low to form a coiled-coil dimer, probably because of insufficient continuity of the hydrophobic residues at (a and d) core positions in amphipathic heptad repeats. On the other hand, several peptides with leucine residues introduced at core positions or with extensional sequences with high alpha helicity had an advantage in coiled-coil formation. When we analyzed the thermal and urea-induced unfolding of these dimeric peptides, we identified four domains having a relatively high potential to form coiled coils. Among them, three domains on the C-terminal side of the stalk region, i.e., (252-272), (276-330), and (336-346), were in the same heptad frame, although these potential coiled-coil domains were not self-sustaining individually. This is in sharp contrast to the fragment of human kinesin, (332-369), which has an extremely high tendency toward coiled-coil formation. One of the possible triggers for coiled-coil formation of the ncd stalk region may be the interaction between the motor domain and the C-terminal part of the stalk as previously revealed by X-ray crystallography. The residues, S331 and R335, seem to act as a breaking point for alpha-helix continuity. This would make the region (336-346), as the head-stalk joint, more flexible such as seen with a plus-end-directed kinesin, if this region had no interaction with the motor domain. These characteristic differences between ncd and kinesin suggest that the nonlocally sustained coiled coil of ncd is one of the factors important for minus-end-directed motility.
Assuntos
Proteínas de Drosophila/química , Cinesinas/química , Sequência de Aminoácidos , Dicroísmo Circular , Humanos , Cinética , Modelos Biológicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Solventes/metabolismo , Temperatura , Termodinâmica , Ureia/metabolismo , Ureia/farmacologiaRESUMO
Mechanisms for the atria-specific action potential-prolonging action of NIP-142 ((3R*,4S*)-4-cyclopropylamino-3,4-dihydro-2,2-dimethyl-6-(4-methoxyphenylacetylamino)-7-nitro-2H-1-benzopyran-3-ol), a benzopyran compound that terminates experimental atrial arrhythmia, was examined. In isolated guinea-pig atrial tissue, NIP-142 reversed the shortening of action potential duration induced by either carbachol or adenosine. These effects were mimicked by tertiapin, but not by E-4031. NIP-142 concentration-dependently blocked the human G protein-coupled inwardly rectifying potassium channel current (GIRK1/4 channel current) expressed in HEK-293 cells with an EC50 value of 0.64 microM. At higher concentrations, NIP-142 blocked the human ether a go-go related gene (HERG) channel current with an EC50 value of 44 microM. In isolated guinea-pig papillary muscles, NIP-142 had no effect on the negative inotropic effect of carbachol under beta-adrenergic stimulation, indicating lack of effect on the muscarinic receptor and Gi protein. These results suggest that NIP-142 directly inhibits the acetylcholine-activated potassium current.
Assuntos
Antiarrítmicos/farmacologia , Função Atrial/efeitos dos fármacos , Benzopiranos/farmacologia , Carbacol/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Potenciais de Ação/efeitos dos fármacos , Adenosina/farmacologia , Animais , Função Atrial/fisiologia , Atropina/farmacologia , Venenos de Abelha/farmacologia , Carbacol/antagonistas & inibidores , Linhagem Celular , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/fisiologia , Cobaias , Humanos , Técnicas In Vitro , Contração Miocárdica/efeitos dos fármacos , Músculos Papilares/efeitos dos fármacos , Músculos Papilares/fisiologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Fatores de TempoRESUMO
NIP-142 is a novel benzopyran compound that was shown to prolong the atrial effective refractory period and terminate experimental atrial fibrillation in the dog. In the present study, we examined the effects of NIP-142 on isolated guinea pig myocardium and on the G-protein-coupled inwardly rectifying potassium channel current (acetylcholine-activated potassium current; I(KACh)) expressed in Xenopus oocytes. NIP-142 (10 and 100 microM) concentration-dependently prolonged the refractory period and action potential duration in the atrium but not in the ventricle. E-4031 and 4-aminopyridine prolonged action potential duration in both left atrium and right ventricle. Prolongation by NIP-142 of the atrial action potential duration was observed at stimulation frequencies between 0.5 and 5 Hz. In contrast, the prolongation by E-4031 was not observed at higher frequencies. Tertiapin, a blocker of I(KACh), prolonged action potential duration in the atrium but not in the ventricle. NIP-142 completely reversed the carbachol-induced shortening of atrial action potential duration. NIP-142 (1 to 100 microM), as well as tertiapin (0.1 to 100 nM), concentration-dependently blocked I(KACh) expressed in Xenopus oocytes; the blockade by NIP-142 was not affected by membrane voltage. In conclusion, NIP-142 was shown to prolong atrial refractory period and action potential duration through blockade of I(KACh) which may possibly explain its previously described antiarrhythmic activity. NIP-142 has pharmacological properties that are different from classical class III antiarrhythmic agents such as atria specificity and lack of reverse frequency dependence, and thus appears promising for the treatment of supraventricular arrhythmia.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Antiarrítmicos/farmacologia , Benzopiranos/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miocárdio , Potenciais de Ação/fisiologia , Animais , Benzopiranos/química , Relação Dose-Resposta a Droga , Feminino , Cobaias , Átrios do Coração/efeitos dos fármacos , Humanos , Técnicas In Vitro , Contração Miocárdica/fisiologia , Xenopus laevisRESUMO
WW domains are universal protein modules for binding Pro-rich ligands. They are classified into four groups according to their binding specificity. Arg-14 and Arg-17, on the WW domain of Pin1, are thought to be important for the binding of Group IV ligands that have (Ser(P)/Thr(P))-Pro sequences. We have applied surface plasmon resonance to determine the ligand specificity of several WW domains containing Arg-14. Among these WW domains, Rsp5.2 and mNedd4.3 bound only to the Group I ligand containing Pro-Pro-Xaa-Tyr with K(D) values of 11 and 55 microm, respectively. The WW domains of hPin1, Caenorhabditis elegans Pin1 homologue (Y110), PinA, and SspI bound to Group IV ligands with K(D) values ranging from 22 to 700 microm. PinA and SspI do not have Arg-17, unlike Pin1 and Y110. The modeled structures of the WW domains of PinA and SspI revealed that the structure and the network of hydrogen bonds of Loop I, which are also formed in Pin1 and Y110, are conserved. We propose that this configuration of Loop I (referred to as the "p patch") is necessary for binding Group IV ligands and that it can be used to predict the specificity and functions of other WW domains.
Assuntos
Proteínas de Choque Térmico HSP70 , Peptidilprolil Isomerase/química , Proteínas de Schizosaccharomyces pombe , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Arginina/química , Sítios de Ligação , Caenorhabditis elegans/metabolismo , Ligação de Hidrogênio , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Peptidilprolil Isomerase de Interação com NIMA , Biossíntese Peptídica , Peptidilprolil Isomerase/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Quinases/química , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
Human LECT2 is a 16-kDa chemotactic protein that consists of 133 amino acids and three intramolecular disulfide bonds. Here, we present the oxidative refolding of (His)(6)-LECT2, an N-terminally (His)(6)-tagged recombinant protein of human LECT2. (His)(6)-LECT2 was overproduced in Escherichia coli in the form of insoluble aggregates, solubilized with 8 M urea in the presence of 10 mM DTT, and purified and refolded on Ni-NTA agarose by lowering the urea concentration before the elution. This process, however, gave a mixture of oligomers of (His)(6)-LECT2 as well as the monomer, whose composition was as low as 36%. The oligomers formed as a result of incorrect intermolecular disulfide bonds. After the refolding on Ni-NTA agarose (step 1), the disulfide bonds were shuffled using a glutathione redox buffer (step 2) and the remaining thiols were completely oxidized (step 3) to improve the yield of correctly folded, monomeric (His)(6)-LECT2. The monomer composition was significantly improved to 81% by the three-step refolding method and the monomer thus obtained was shown to have the same conformation as the authentic LECT2 produced in CHO cells by CD and NMR spectroscopies. The yield of (His)(6)-LECT2 was 1.0 mg/L E. coli culture and was 16 times as high as that in our previous report, in which (His)(6)-LECT2 was purified from the soluble fractions of E. coli cell lysates.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Oxigênio/metabolismo , Proteínas/química , Animais , Células CHO , Dicroísmo Circular , Cricetinae , Dissulfetos/química , Ditiotreitol/farmacologia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Oxirredução , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/metabolismo , Sefarose/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria , Fatores de Tempo , Ureia/farmacologiaRESUMO
The effects of terfenadine, an antiallergic drug also known for its QT-prolonging and arrhythmogenic activities, on the action potential of isolated myocardial tissue preparations from rabbits were examined with microelectrode techniques. In the Purkinje fibers and atrium, terfenadine concentration dependently decreased the maximum rate of rise (+.V(max)) without affecting other action potential parameters. In the ventricle, terfenadine had little effect on action potential configuration. In the sinoatrial node, terfenadine 20 microM prolonged cycle length mainly through inhibition of +.V(max). Terfenadine 1 microM completely inhibited the human ether a go-go-related gene (HERG) channel current expressed in HEK293 cells in the same experimental solution as in microelectrode experiments. The lack of terfenadine effect on the action potential duration suggests that there are drugs for which the HERG channel inhibitory action underlying in vivo QT prolongation cannot be evaluated based on their action potential-prolonging activity in isolated myocardial tissue preparations.