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1.
World J Microbiol Biotechnol ; 35(5): 77, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31069553

RESUMO

Ethylene is a volatile alkene which is used in large commercial scale as a precursor in plastic industry, and is currently derived from petroleum refinement. As an alternative production strategy, photoautotrophic cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have been previously evaluated as potential biotechnological hosts for producing ethylene directly from CO2, by the over-expression of ethylene forming enzyme (efe) from Pseudomonas syringae. This work addresses various open questions related to the use of Synechococcus as the engineering target, and demonstrates long-term ethylene production at rates reaching 140 µL L-1 h-1 OD750-1 without loss of host vitality or capacity to produce ethylene. The results imply that the genetic instability observed earlier may be associated with the expression strategies, rather than efe over-expression, ethylene toxicity or the depletion of 2-oxoglutarate-derived cellular precursors in Synechococcus. In context with literature, this study underlines the critical differences in expression system design in the alternative hosts, and confirms Synechococcus as a suitable parallel host for further engineering.


Assuntos
Etilenos/biossíntese , Engenharia Metabólica/métodos , Fotossíntese/fisiologia , Synechococcus/genética , Synechococcus/metabolismo , Biotecnologia , Dióxido de Carbono/metabolismo , Clonagem Molecular , Tolerância a Medicamentos , Escherichia coli/genética , Etilenos/toxicidade , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Instabilidade Genômica , Ácidos Cetoglutáricos/metabolismo , Liases/genética , Liases/metabolismo , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Synechococcus/efeitos dos fármacos , Synechococcus/crescimento & desenvolvimento , Transformação Genética
2.
Res Vet Sci ; 124: 393-398, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31077967

RESUMO

Osteoarthritis associated with hip dysplasia is one of the most common orthopedic abnormalities in dogs, with an incidence of up to 40% in some breeds. Tissue therapy of cartilage has received great attention, with use of mesenchymal stromal cells and different types of biomaterials. The present study aimed to evaluate the effect of platelet lysate (PL) on the proliferation and differentiation of canine adipose tissue-derived mesenchymal stromal cells (ASCs), in liquid culture or hydrogels. PL was prepared from blood collected from healthy dogs and submitted to freezing-thawing cycles, and hydrogel was formed with canine thrombin. The effect of PL on the proliferation and differentiation of canine ASCs was evaluated in liquid and hydrogel systems, with microscopy, quantification of dsDNA, histology and quantification of glycosaminoglycans. The addition of 5% or 10% PL to the culture medium induced a greater proliferation rate than the presence of 10% fetal bovine serum. The cultivation of ASCs in PL gel, with normal or chondrogenic medium, resulted in maintenance of proliferation level similar to the conventional 2D cultivation, and induction of chondrogenic differentiation, especially in the presence of the chondrogenesis induction medium.


Assuntos
Tecido Adiposo/fisiologia , Condrogênese/fisiologia , Liases/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Plaquetas/enzimologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Liases/administração & dosagem , Células-Tronco Mesenquimais/fisiologia
3.
Phys Chem Chem Phys ; 21(19): 9957-9968, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31041955

RESUMO

The ethylene-forming enzyme (EFE) is a unique member of the Fe(ii)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases. It converts 2OG into ethylene plus three CO2 molecules (ethylene-forming reaction) and also catalyzes the C5 hydroxylation of l-arginine coupled to the oxidative decarboxylation of 2OG (l-Arg hydroxylation reaction). To uncover the mechanisms of the dual transformations by EFE, quantum mechanical/molecular mechanical (QM/MM) calculations were carried out. Based on the results, a branched mechanism was proposed. An FeII-peroxysuccinate complex with a dissociated CO2 generated through the nucleophilic attack of the superoxo moiety of the Fe-O2 species on the keto carbon of 2OG is the key common intermediate in both reactions. A competition between the subsequent CO2 insertion (a key step in the ethylene-forming pathway) and the O-O bond cleavage (leading to the formation of succinate) governs the product selectivity. The calculated reaction barriers suggested that the CO2 insertion is favored over the O-O bond cleavage. This is consistent with the product preference observed in experiments. By comparison with the results of AsqJ (an Fe/2OG oxygenase that leads to substrate oxidation exclusively), the protein environment was found to be crucial for the selectivity. Further calculations demonstrated that the local electric field of the protein environment in EFE promotes ethylene formation by acting as a charge template, exemplifying the importance of the electrostatic interaction in enzyme catalysis. These findings offer mechanistic insights into the EFE catalysis and provide important clues for better understanding the unique ethylene-forming capability of EFE compared with other Fe/2OG oxygenases.


Assuntos
Arginina/metabolismo , Etilenos/biossíntese , Ácidos Cetoglutáricos/metabolismo , Liases/metabolismo , Arginina/química , Biocatálise , Teoria da Densidade Funcional , Etilenos/química , Hidroxilação , Ácidos Cetoglutáricos/química , Liases/química , Estrutura Molecular , Oxirredução
4.
Nat Med ; 25(4): 667-678, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936548

RESUMO

Several studies have investigated links between the gut microbiome and colorectal cancer (CRC), but questions remain about the replicability of biomarkers across cohorts and populations. We performed a meta-analysis of five publicly available datasets and two new cohorts and validated the findings on two additional cohorts, considering in total 969 fecal metagenomes. Unlike microbiome shifts associated with gastrointestinal syndromes, the gut microbiome in CRC showed reproducibly higher richness than controls (P < 0.01), partially due to expansions of species typically derived from the oral cavity. Meta-analysis of the microbiome functional potential identified gluconeogenesis and the putrefaction and fermentation pathways as being associated with CRC, whereas the stachyose and starch degradation pathways were associated with controls. Predictive microbiome signatures for CRC trained on multiple datasets showed consistently high accuracy in datasets not considered for model training and independent validation cohorts (average area under the curve, 0.84). Pooled analysis of raw metagenomes showed that the choline trimethylamine-lyase gene was overabundant in CRC (P = 0.001), identifying a relationship between microbiome choline metabolism and CRC. The combined analysis of heterogeneous CRC cohorts thus identified reproducible microbiome biomarkers and accurate disease-predictive models that can form the basis for clinical prognostic tests and hypothesis-driven mechanistic studies.


Assuntos
Colina/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/microbiologia , Metagenômica , Biomarcadores Tumorais/metabolismo , Estudos de Coortes , Neoplasias Colorretais/diagnóstico , Bases de Dados Genéticas , Microbioma Gastrointestinal , Humanos , Liases/genética , Liases/metabolismo , Especificidade da Espécie
5.
Plant Sci ; 280: 321-329, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30824011

RESUMO

Cysteine functions not only as an amino acid in proteins but also as a precursor for a large number of essential biomolecules. Cysteine is synthesized via the incorporation of sulfide to O-acetylserine under the catalysis of O-acetylserine(thiol)lyase (OASTL). In dicotyledonous Arabidopsis, nine OASTL genes have been reported. However, in their null mutants, only the mutant of CS26 encoding S-sulfocysteine synthase showed the visible phenotypic changes, displaying significantly small plants and pale-green leaves under long-day condition but not short-day condition. Up to now, no OASTL gene or mutant has been identified in monocotyledon. In this study, we isolated a green-revertible albino mutant gra78 in rice (Oryza sativa). Its albino phenotype at the early seedling stage was sensitive to temperature but independent of photoperiod. Map-based cloning revealed that candidate gene LOC_Os01g59920 of GRA78 encodes a putative S-sulfocysteine synthase showing significant similarity with Arabidopsis CS26. Complementation experiment confirmed that mutation in LOC_Os01g59920 accounted for the mutant phenotype of gra78. GRA78 is constitutively expressed in all tissues and its encoded protein is targeted to the chloroplast. In addition, qRT-PCR suggested that expression levels of four OASTL homolog genes and five photosynthetic genes were remarkably down-regulated.


Assuntos
Liases/metabolismo , Oryza/enzimologia , Cloroplastos/fisiologia , Cloroplastos/ultraestrutura , Liases/genética , Liases/ultraestrutura , Mutação , Oryza/genética , Oryza/crescimento & desenvolvimento , Oryza/ultraestrutura , Fenótipo , Fotossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/ultraestrutura
6.
Proc Natl Acad Sci U S A ; 116(13): 6457-6462, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30846551

RESUMO

Marine Synechococcus, a globally important group of cyanobacteria, thrives in various light niches in part due to its varied photosynthetic light-harvesting pigments. Many Synechococcus strains use a process known as chromatic acclimation to optimize the ratio of two chromophores, green-light-absorbing phycoerythrobilin (PEB) and blue-light-absorbing phycourobilin (PUB), within their light-harvesting complexes. A full mechanistic understanding of how Synechococcus cells tune their PEB to PUB ratio during chromatic acclimation has not yet been obtained. Here, we show that interplay between two enzymes named MpeY and MpeZ controls differential PEB and PUB covalent attachment to the same cysteine residue. MpeY attaches PEB to the light-harvesting protein MpeA in green light, while MpeZ attaches PUB to MpeA in blue light. We demonstrate that the ratio of mpeY to mpeZ mRNA determines if PEB or PUB is attached. Additionally, strains encoding only MpeY or MpeZ do not acclimate. Examination of strains of Synechococcus isolated from across the globe indicates that the interplay between MpeY and MpeZ uncovered here is a critical feature of chromatic acclimation for marine Synechococcus worldwide.


Assuntos
Aclimatação/fisiologia , Aclimatação/efeitos da radiação , Adaptação Ocular/fisiologia , Adaptação Ocular/efeitos da radiação , Cor , Synechococcus/enzimologia , Synechococcus/metabolismo , Aclimatação/genética , Adaptação Ocular/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica , Genes Bacterianos/genética , Liases/metabolismo , Mutação , Ficobilinas , Ficoeritrina , Proteínas Recombinantes , Água do Mar/microbiologia , Synechococcus/genética , Synechococcus/efeitos da radiação , Urobilina/análogos & derivados
7.
Plant Biol (Stuttg) ; 21(4): 595-603, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30734982

RESUMO

Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1-aminocyclopropane-1-carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO-associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence. Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography-tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms. Our observations showed that the loss-of-function acs1-1 mutant ameliorated age- or dark-induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1-1 reduced ACC accumulation mainly in mature leaves and that acs1-1-promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO-deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence. These findings suggest that NOA1-dependent NO accumulation blocked the ACS1-induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.


Assuntos
Aminoácidos Cíclicos/metabolismo , Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Liases/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismo , Glucuronidase/metabolismo , Liases/fisiologia , Óxido Nítrico Sintase/fisiologia , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Transcriptoma
8.
Org Biomol Chem ; 17(2): 248-251, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30548032

RESUMO

A putative three-gene cluster for asperterpenoid A was identified. Step-wise reconstitution of this gene cluster in Aspergillus oryzae reveals that astC encodes a sesterterpene cyclase to synthesize preasperterpenoid A, which is dually oxidized by a P450 enzyme AstB to give asperterpenoid A along with a minor product asperterpenoid B, and asperterpenoid A is further oxidized by another P450 eznyme AstA to afford a new sesterterpenoid asperterpenoid C. Unexpectedly, asperterpenoids A and B, but not the final product asperterpenoid C, exhibit potent inhibitory activity against Mycobacterium tuberculosis protein tyrosine phosphatase B with IC50 values of 3-6 µM.


Assuntos
Antituberculosos/metabolismo , Antituberculosos/farmacologia , Aspergillus oryzae/metabolismo , Mycobacterium tuberculosis/enzimologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacologia , Aspergillus oryzae/enzimologia , Aspergillus oryzae/genética , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Liases/metabolismo , Família Multigênica , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico
9.
Plant Mol Biol ; 99(1-2): 123-134, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30535734

RESUMO

KEY MESSAGE: This research demonstrated the conservation and diversification of the functions of the O-acetylserine-(thiol) lyase gene family genes in Solanum lycopersicum L. Cysteine is the first sulfur-containing organic molecule generated by plants and is the precursor of many important biomolecules and defense compounds. Cysteine and its derivatives are also essential in various redox signaling-related processes. O-acetylserine(thiol)lyase (OASTL) proteins catalyze the last step of cysteine biosynthesis. Previously, researches focused mainly on OASTL proteins which were the most abundant or possessed the authentic OASTL activity, whereas few studies have ever given a comprehensive view of the functions of all the OASTL members in one specific species. Here, we characterized 8 genes belonging to the OASTL gene family from tomato genome (SlOAS2 to SlOAS9), including the sequence analyses, subcellular localization, enzymatic activity assays, expression patterns, as well as the interaction property with SATs. Apart from SlOAS3, all the other genes encoded OASTL-like proteins. Tomato OASTLs were differentially expressed during the development of tomato plants, and their encoded proteins had diverse compartmental distributions and functions. SlOAS5 and SlOAS6 catalyzed the biogenesis of cysteine in chloroplasts and in the cytosol, respectively, and this was in consistent with their interaction abilities with SlSATs. SlOAS4 catalyzed the generation of hydrogen sulfide, similar to its Arabidopsis ortholog, DES1. SlOAS2 also functioned as an L-cysteine desulfhydrase, but its expression pattern was very different from that of SlOAS4. Additionally, SlOAS8 might be a ß-cyanoalanine synthase in mitochondria, and the S-sulfocysteine synthase activity appeared lost in tomato plants. SlOAS7 exhibited a transactivational ability in yeast; while the subcellular localization of SlOAS9 was in the peroxisome and correlated with the process of leaf senescence, indicating that these two genes might have novel roles.


Assuntos
Carbono-Oxigênio Liases/genética , Lycopersicon esculentum/enzimologia , Família Multigênica , Carbono-Oxigênio Liases/metabolismo , Cloroplastos/metabolismo , Cisteína/metabolismo , Citosol/metabolismo , Liases/genética , Liases/metabolismo , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
10.
Org Lett ; 20(17): 5427-5430, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30141637

RESUMO

Ovothiols are thiolhistidine derivatives. The first step of ovothiol biosynthesis is OvoA-catalyzed oxidative coupling between histidine and cysteine. In this report, the remaining steps of ovothiol A biosynthesis were reconstituted in vitro. ETA_14770 (OvoB) was reported as a PLP-dependent sulfoxide lyase, responsible for mercaptohistidine production. OvoA was found to be a bifunctional enzyme, which mediates both oxidative C-S bond formation and methylation of mercaptohistidine to afford ovothiol A. Besides reconstituting the whole biosynthetic pathway, two unique features proposed in the literature were also examined: a potential cysteine-recycling mechanism of the C-S lyase (OvoB) and the selectivity of the π- N methyltransferase.


Assuntos
Liases/metabolismo , Metilistidinas/metabolismo , Metiltransferases/metabolismo , Liases/química , Metilistidinas/química , Metiltransferases/química , Modelos Moleculares , Conformação Proteica
11.
Molecules ; 23(8)2018 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-30042370

RESUMO

Ferns are the most primitive of all vascular plants. One of the characteristics distinguishing them from flowering plants is its triterpene metabolism. Most cyclic triterpenes in ferns are hydrocarbons derived from the direct cyclization of squalene by squalene cyclases (SCs). Both ferns and more complex plants share sterols and biosynthetic enzymes, such as cycloartenol synthases (CASs). Polystichum belongs to Dryopteridaceae, and is one of the most species-rich of all fern genera. Several Polystichum ferns in Japan are classified as one of three possible chemotypes, based on their triterpene profiles. In this study, we describe the molecular cloning and functional characterization of cDNAs encoding a SC (PPH) and a CAS (PPX) from the type species Polystichum polyblepharum. Heterologous expression in Pichia pastoris revealed that PPH and PPX are hydroxyhopane synthase and CAS, respectively. By using the PPH and PPX sequences, we successfully isolated SC- and CAS-encoding cDNAs from six Polystichum ferns. Phylogenetic analysis, based on SCs and oxidosqualene cyclase sequences, suggested that the Polystichum subclade in the fern SC and CAS clades reflects the chemotype-but not the molecular phylogeny constructed using plastid molecular markers. These results show a possible relation between triterpenes and their biosynthetic enzymes in Polystichum.


Assuntos
Transferases Intramoleculares/genética , Liases/genética , Proteínas de Plantas/genética , Plastídeos/genética , Polystichum/genética , Triterpenos/metabolismo , Sequência de Aminoácidos , Clonagem Molecular/métodos , DNA Complementar/genética , DNA Complementar/metabolismo , Expressão Gênica , Marcadores Genéticos , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Transferases Intramoleculares/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Japão , Liases/metabolismo , Filogenia , Pichia/enzimologia , Pichia/genética , Proteínas de Plantas/metabolismo , Plastídeos/enzimologia , Polystichum/classificação , Polystichum/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Triterpenos/química , Triterpenos/isolamento & purificação
12.
Eur J Med Chem ; 155: 754-763, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29940465

RESUMO

We report on the virtual screening, synthesis, and biological evaluation of new furan derivatives targeting Mycobacterium tuberculosis salicylate synthase (MbtI). A receptor-based virtual screening procedure was applied to screen the Enamine database, identifying two compounds, I and III, endowed with a good enzyme inhibitory activity. Considering the most active compound I as starting point for the development of novel MbtI inhibitors, we obtained new derivatives based on the furan scaffold. Among the SAR performed on this class, compound 1a emerged as the most potent MbtI inhibitor reported to date (Ki = 5.3 µM). Moreover, compound 1a showed a promising antimycobacterial activity (MIC99 = 156 µM), which is conceivably related to mycobactin biosynthesis inhibition.


Assuntos
Antituberculosos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Liases/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/síntese química , Antituberculosos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Liases/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Relação Estrutura-Atividade
13.
Molecules ; 23(7)2018 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-29933627

RESUMO

Tuberculosis is the leading cause of death from a single infectious agent worldwide; therefore, the need for new antitubercular drugs is desperate. The recently validated target salicylate synthase MbtI is the first enzyme involved in the biosynthesis of mycobactins, compounds able to chelate iron, an essential cofactor for the survival of Mycobacterium tuberculosis in the host. Here, we report on the synthesis and biological evaluation of chromane-based compounds as new potential inhibitors of MbtI. Our approach successfully allowed the identification of a novel lead compound (1), endowed with a promising activity against this enzyme (IC50 = 55 μM). Molecular modeling studies were performed in order to evaluate the binding mode of 1 and rationalize the preliminary structure-activity relationships, thus providing crucial information to carry out further optimization studies.


Assuntos
Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Ácido Corísmico/química , Cromanos/química , Inibidores Enzimáticos/química , Liases/antagonistas & inibidores , Mycobacterium tuberculosis/química , Motivos de Aminoácidos , Antituberculosos/síntese química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Ácido Corísmico/metabolismo , Cromanos/síntese química , Inibidores Enzimáticos/síntese química , Expressão Gênica , Cinética , Liases/química , Liases/genética , Liases/metabolismo , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/enzimologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Termodinâmica
14.
Proc Natl Acad Sci U S A ; 115(21): E4870-E4879, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29735649

RESUMO

Capsules are surface layers of hydrated capsular polysaccharides (CPSs) produced by many bacteria. The human pathogen Salmonella enterica serovar Typhi produces "Vi antigen" CPS, which contributes to virulence. In a conserved strategy used by bacteria with diverse CPS structures, translocation of Vi antigen to the cell surface is driven by an ATP-binding cassette (ABC) transporter. These transporters are engaged in heterooligomeric complexes proposed to form an enclosed translocation conduit to the cell surface, allowing the transporter to power the entire process. We identified Vi antigen biosynthesis genetic loci in genera of the Burkholderiales, which are paradoxically distinguished from S. Typhi by encoding VexL, a predicted pectate lyase homolog. Biochemical analyses demonstrated that VexL is an unusual metal-independent endolyase with an acidic pH optimum that is specific for O-acetylated Vi antigen. A 1.22-Å crystal structure of the VexL-Vi antigen complex revealed features which distinguish common secreted catabolic pectate lyases from periplasmic VexL, which participates in cell-surface assembly. VexL possesses a right-handed parallel ß-superhelix, of which one face forms an electropositive glycan-binding groove with an extensive hydrogen bonding network that includes Vi antigen acetyl groups and confers substrate specificity. VexL provided a probe to interrogate conserved features of the ABC transporter-dependent export model. When introduced into S Typhi, VexL localized to the periplasm and degraded Vi antigen. In contrast, a cytosolic derivative had no effect unless export was disrupted. These data provide evidence that CPS assembled in ABC transporter-dependent systems is actually exposed to the periplasm during envelope translocation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Burkholderia/enzimologia , Liases/metabolismo , Periplasma/enzimologia , Polissacarídeos Bacterianos/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Transporte Biológico , Liases/química , Filogenia , Conformação Proteica
15.
Appl Microbiol Biotechnol ; 102(13): 5391-5401, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29728724

RESUMO

Lignin is the major aromatic biopolymer in nature, and it is considered a valuable feedstock for the future supply of aromatics. Hence, its valorisation in biorefineries is of high importance, and various chemical and enzymatic approaches for lignin depolymerisation have been reported. Among the enzymes known to act on lignin, ß-etherases offer the possibility for a selective cleavage of the ß-O-4 aryl ether bonds present in lignin. These enzymes, together with glutathione lyases, catalyse a reductive, glutathione-dependent ether bond cleavage displaying high stereospecificity. ß-Etherases and glutathione lyases both belong to the superfamily of glutathione transferases, and several structures have been solved recently. Additionally, different approaches for their application in lignin valorisation have been reported in the last years. This review gives an overview on the current knowledge on ß-etherases and glutathione lyases, their biochemical and structural features, and critically discusses their potential for application in biorefineries.


Assuntos
Proteínas de Bactérias/metabolismo , Reatores Biológicos , Lignina/metabolismo , Liases/metabolismo , Oxirredutases/metabolismo
16.
Plant Cell Physiol ; 59(5): 1072-1083, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29490083

RESUMO

In Arabidopsis thaliana, cyanide is produced concomitantly with ethylene biosynthesis and is mainly detoxified by the ß-cyanoalanine synthase CAS-C1. In roots, CAS-C1 activity is essential to maintain a low level of cyanide for proper root hair development. Root hair elongation relies on polarized cell expansion at the growing tip, and we have observed that CAS-C1 locates in mitochondria and accumulates in root hair tips during root hair elongation, as shown by observing the fluorescence in plants transformed with the translational construct ProC1:CASC1-GFP, containing the complete CAS-C1 gene fused to green fluorescent protein (GFP). Mutants in the SUPERCENTIPEDE (SCN1) gene, that regulate the NADPH oxidase gene ROOT HAIR DEFECTIVE 2 (RHD2)/AtrbohC, are affected at the very early steps of the development of root hair that do not elongate and do not show a preferential localization of the GFP accumulation in the tips of the root hair primordia. Root hairs of mutants in CAS-C1 or RHD2/AtrbohC, whose protein product catalyzes the generation of ROS and the Ca2+ gradient, start to grow out correctly, but they do not elongate. Genetic crosses between the cas-c1 mutant and scn1 or rhd2 mutants were performed, and the detailed phenotypic and molecular characterization of the double mutants demonstrates that scn1 mutation is epistatic to cas-c1 and cas-c1 is epistatic to rhd2 mutation, indicating that CAS-C1 acts in early steps of the root hair development process. Moreover, our results show that the role of CAS-C1 in root hair elongation is independent of H2O2 production and of a direct NADPH oxidase inhibition by cyanide.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Cianetos/toxicidade , Cisteína Sintase/metabolismo , Liases/metabolismo , NADPH Oxidases/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Trifosfato de Adenosina/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Cisteína Sintase/antagonistas & inibidores , Cisteína Sintase/genética , Ativação Enzimática/efeitos dos fármacos , Epistasia Genética/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Hidroxocobalamina/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Mutação/genética , NADPH Oxidases/antagonistas & inibidores , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Superóxidos/metabolismo
17.
Trends Biochem Sci ; 43(5): 342-357, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29573882

RESUMO

The menaquinone, siderophore, and tryptophan (MST) enzymes transform chorismate to generate precursor molecules for the biosynthetic pathways defined in their name. Kinetic data, both steady-state and transient-state, and X-ray crystal structures indicate that these enzymes are highly conserved both in mechanism and in structure. Because these enzymes are found in pathogens but not in humans, there is considerable interest in these enzymes as drug design targets. While great progress has been made in defining enzyme structure and mechanism, inhibitor design has lagged behind. This review provides a detailed description of the evidence that begins to unravel the mystery of how the MST enzymes work, and how that information has been used in inhibitor design.


Assuntos
Liases/metabolismo , Sideróforos/metabolismo , Triptofano/metabolismo , Vitamina K 2/metabolismo , Humanos , Cinética , Liases/química , Modelos Moleculares , Sideróforos/química , Triptofano/química , Vitamina K 2/química
18.
Biochem Biophys Res Commun ; 497(2): 749-755, 2018 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-29462612

RESUMO

Mg chelatase, a key enzyme in chlorophyll biosynthesis, is comprised of I, D and H subunits. Among these subunits, the D subunit was regarded to mediate protein interactions due to its unique protein domains. However, the functional roles of the different domains of the D subunit in vivo remain unclear. In this study, we dissected the rice (Oryza sativa) D subunit (OsCHLD) into three peptide fragments: the putative chloroplast transit peptide (TP, Met1 to Arg45), the N-terminus plus linker domain (OsCHLDN + L, Ala46 to Leu485) and the C-terminus (OsCHLDC, Ile486 to Ser754), to explore the roles of these fragments. The results of the yeast two-hybrid assay and the in vitro reconstitution of the Mg-chelatase activity showed that only OsCHLDN + L interacted with the I and H subunits and maintained most of the Mg-chelatase activity in vitro. Furthermore, artificial TP-OsCHLDN + L and TP-OsCHLDC were overexpressed in rice. Interestingly, an incomplete co-suppression had occurred in both of the overexpressed (OsCHLDN + L-ox and OsCHLDC-ox) plants, resulting in a significantly downregulated expression of endogenous OsCHLD. Therefore, these transgenic plants had adequate OsCHLDN + L and OsCHLDC instead of endogenous OsCHLD, providing ideal models to study the function of different domains of the D subunit in vivo. The OsCHLDN + L-ox plants showed an identical phenotype to that of the wild type, while the OsCHLDC-ox plants demonstrated a yellowish phenotype that resembled the D subunit mutants. These results indicated that only OsCHLDN + L could complement the function of endogenous OsCHLD, providing direct evidence that OsCHLDN + L is essential for Mg-chelatase activity in vivo.


Assuntos
Liases/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Cloroplastos/química , Cloroplastos/genética , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Regulação da Expressão Gênica de Plantas , Liases/química , Liases/genética , Oryza/química , Oryza/genética , Oryza/ultraestrutura , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/ultraestrutura , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Regulação para Cima
19.
J Proteome Res ; 17(3): 1290-1299, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29405720

RESUMO

Nutrient starvation is an important survival challenge for bacteria during industrial production of functional foods. As next-generation sequencing technology has greatly advanced, we performed proteomic and genomic analysis to investigate the response of Lactobacillus casei Zhang to a glucose-restricted environment. L. casei Zhang strains were permitted to evolve in glucose-restricted or normal medium from a common ancestor over a 3 year period, and they were sampled at 1000, 2000, 3000, 4000, 5000, 6000, 7000, and 8000 generations and subjected to proteomic and genomic analyses. Genomic resequencing data revealed different point mutations and other mutational events in each selected generation of L. casei Zhang under glucose restriction stress. The differentially expressed proteins induced by glucose restriction were mostly related to fructose and mannose metabolism, carbohydrate metabolic processes, lyase activity, and amino-acid-transporting ATPase activity. Integrative proteomic and genomic analysis revealed that the mutations protected L. casei Zhang against glucose starvation by regulating other cellular carbohydrate, fatty acid, and amino acid catabolism; phosphoenolpyruvate system pathway activation; glycogen synthesis; ATP consumption; pyruvate metabolism; and general stress-response protein expression. The results help reveal the mechanisms of adapting to glucose starvation and provide new strategies for enhancing the industrial utility of L. casei Zhang.


Assuntos
Adaptação Fisiológica/genética , Genoma Bacteriano , Glucose/deficiência , Lactobacillus casei/metabolismo , Proteômica/métodos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Aminoácidos/metabolismo , Transporte Biológico , Meios de Cultura/química , Meios de Cultura/farmacologia , Ácidos Graxos/metabolismo , Frutose/metabolismo , Expressão Gênica , Glucose/farmacologia , Glicogênio/biossíntese , Lactobacillus casei/efeitos dos fármacos , Lactobacillus casei/genética , Lactobacillus casei/crescimento & desenvolvimento , Liases/genética , Liases/metabolismo , Manose/metabolismo , Fosfoenolpiruvato/metabolismo , Mutação Puntual , Estresse Fisiológico
20.
Bioorg Med Chem ; 26(7): 1275-1284, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28709846

RESUMO

Biocatalysis has been increasingly used for pharmaceutical synthesis in an effort to make manufacturing processes greener and more sustainable. Biocatalysts that possess excellent activity, specificity, thermostability and solvent-tolerance are highly sought after to meet the requirements of practical applications. Generating biocatalysts with these specific properties can be achieved by either discovery of novel biocatalysts or protein engineering. Meanwhile, chemoenzymatic routes have also been designed and developed for pharmaceutical synthesis on an industrial scale. This review discusses the recent discoveries, engineering, and applications of biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. Key classes of biocatalysts include reductases, oxidases, hydrolases, lyases, isomerases, and transaminases.


Assuntos
Hidrolases/metabolismo , Isomerases/metabolismo , Liases/metabolismo , Oxirredutases/metabolismo , Preparações Farmacêuticas/metabolismo , Transaminases/metabolismo , Biocatálise , Humanos , Preparações Farmacêuticas/química , Engenharia de Proteínas
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