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1.
J Agric Food Chem ; 67(38): 10624-10636, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31483633

RESUMO

The freshness and color quality of postharvest tea leaves can be markedly prolonged and retained by proper preservation measures. Here, we investigated the dynamic changes of chlorophyll and its derivatives in postharvest tea leaves under different low-temperature treatments using natural withering as a control. Chlorophyll decomposition was found closely related with chlorophyllide, pheophorbide, and pheophytin. Low-temperature withering could slow chlorophyll degradation in postharvest tea leaves via significant inhibition on the enzyme activity and gene expression of Mg-dechelatase, chlorophyllase, and pheophorbide a oxygenase. At the initial stage of withering, a significant increase was observed in the chlorophyll content, expression of chlorophyll-synthesis-related enzymes (such as glutamyl-tRNA synthetase, etc.), and chlorophyll synthase activity in newly picked tea leaves. Moreover, an obvious decrease was found in the content of l-glutamate as the foremost precursor substance of chlorophyll synthesis. Hence, our findings revealed that the chlorophyll synthesis reaction was induced by the light-dehydration-stress in the initial withering of tea leaves. This study provides a theoretical basis for exploring preservation technology in actual green tea production.


Assuntos
Camellia sinensis/genética , Camellia sinensis/metabolismo , Clorofila/metabolismo , Manipulação de Alimentos/métodos , Regulação da Expressão Gênica de Plantas , Camellia sinensis/química , Camellia sinensis/crescimento & desenvolvimento , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Clorofila/química , Cor , Enzimas/genética , Enzimas/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Folhas de Planta/química , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Temperatura Ambiente
2.
J Agric Food Chem ; 67(32): 8919-8925, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31334658

RESUMO

Histone deacetylase (HDAC) performs important functions in plant growth and development, including fruit ripening. As a complex biological process, fruit ripening involves the histone acetylation modification of ripening-associated genes. Histone deacetylase genes (HDACs) have been well studied in Arabidopsis and rice, but the biological functions of HDACs in papaya are poorly understood. In the present work, three CpHDACs, belonging to the RPD3/HDA1 subfamily, were identified from papaya and named as CpHDA1, CpHDA2, and CpHDA3. CpHDA1 and CpHDA2 were induced by propylene, while CpHDA3 was propylene-repressed. Moreover, CpHDA3 protein could physically interact with CpERF9 and enhance the transcriptional repression activities of CpERF9 to downstream genes CpPME1, CpPME2 and CpPG5. Histone acetylation levels of CpPME1 and CpPG5 were increased during fruit ripening. Taken together, these results suggested that CpERF9 recruits CpHDA3 to form a histone deacetylase repressor complex to mediate pectin methylesterase and polygalacturonase genes expression during papaya fruit ripening and softening.


Assuntos
Hidrolases de Éster Carboxílico/genética , Carica/metabolismo , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Histona Desacetilases/metabolismo , Proteínas de Plantas/metabolismo , Poligalacturonase/genética , Fatores de Transcrição/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Carica/genética , Carica/crescimento & desenvolvimento , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilases/genética , Proteínas de Plantas/genética , Poligalacturonase/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/genética
3.
Yakugaku Zasshi ; 139(5): 837-844, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31061351

RESUMO

The hydrolysis activity and expression level of carboxylesterase (CES) in skin were compared with liver and intestine in the same individual of beagle dog and cynomolgus monkey, and their aging effects were studied. CES1 isozymes were mainly present in skin of both animals. The dermal hydrolysis activity was about 10 and 40% of hepatic activity in beagle dog and cynomolgus monkey, respectively. In beagle dog, the hydrolysis activity and the expression level of CES isozyme in liver and skin were nearly the same between 2- and 11-year-old individuals. On the other hand, the dermal hydrolase activity was lower in young individual than in old, in contrast to slight increase of hepatic and intestinal activity in old cynomolgus monkey. These differences by aging in cynomolgus monkey were related to the expression of CES1 proteins and their mRNA. Furthermore, mRNA level of human CES was investigated using total RNA of two individuals (63 and 85 years old). The two individuals showed approximately 2-fold higher expression of hCE2 than hCE1 in human skin.


Assuntos
Envelhecimento/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Intestinos/enzimologia , Fígado/enzimologia , Pele/enzimologia , Idoso de 80 Anos ou mais , Animais , Cães , Feminino , Expressão Gênica , Humanos , Hidrólise , Isoenzimas/genética , Isoenzimas/metabolismo , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Nat Commun ; 10(1): 2304, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127100

RESUMO

A central problem in speciation is the origin and mechanisms of reproductive barriers that block gene flow between sympatric populations. Wind-pollinated plant species that flower in synchrony with one another rely on post-pollination interactions to maintain reproductive isolation. In some locations in Mexico, sympatric populations of domesticated maize and annual teosinte grow in intimate associate and flower synchronously, but rarely produce hybrids. This trait is typically conferred by a single haplotype, Teosinte crossing barrier1-s. Here, we show that the Teosinte crossing barrier1-s haplotype contains a pistil-expressed, potential speciation gene, encoding a pectin methylesterase homolog. The modification of the pollen tube cell wall by the pistil, then, is likely a key mechanism for pollen rejection in Zea and may represent a general mechanism for reproductive isolation in grasses.


Assuntos
Hidrolases de Éster Carboxílico/genética , Melhoramento Vegetal , Proteínas de Plantas/genética , Isolamento Reprodutivo , Zea mays/genética , Hidrolases de Éster Carboxílico/metabolismo , Cruzamentos Genéticos , Especiação Genética , Mutação , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Tubo Polínico/genética , Tubo Polínico/metabolismo , Simpatria/genética
5.
Mar Drugs ; 17(5)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117226

RESUMO

MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various N-acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomLI144V and MomLV149A. These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of Pectobacterium carotovorum subsp. carotovorum (Pcc). Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on Pcc-infected Chinese cabbage. The results indicated that MomL and its mutants (MomLL254R, MomLI144V, MomLV149A) significantly decreased the pathogenicity of Pcc. This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase.


Assuntos
Aminoácidos/análise , Hidrolases de Éster Carboxílico , Pectobacterium carotovorum/enzimologia , Pectobacterium carotovorum/genética , Engenharia de Proteínas , Substituição de Aminoácidos , Brassica rapa/microbiologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Ativação Enzimática/genética , Mutação , Pectobacterium carotovorum/patogenicidade , Virulência/genética
6.
Int J Mol Sci ; 20(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018531

RESUMO

The evolution of release factors catalyzing the hydrolysis of the final peptidyl-tRNA bond and the release of the polypeptide from the ribosome has been a longstanding paradox. While the components of the translation apparatus are generally well-conserved across extant life, structurally unrelated release factor peptidyl hydrolases (RF-PHs) emerged in the stems of the bacterial and archaeo-eukaryotic lineages. We analyze the diversification of RF-PH domains within the broader evolutionary framework of the translation apparatus. Thus, we reconstruct the possible state of translation termination in the Last Universal Common Ancestor with possible tRNA-like terminators. Further, evolutionary trajectories of the several auxiliary release factors in ribosome quality control (RQC) and rescue pathways point to multiple independent solutions to this problem and frequent transfers between superkingdoms including the recently characterized ArfT, which is more widely distributed across life than previously appreciated. The eukaryotic RQC system was pieced together from components with disparate provenance, which include the long-sought-after Vms1/ANKZF1 RF-PH of bacterial origin. We also uncover an under-appreciated evolutionary driver of innovation in rescue pathways: effectors deployed in biological conflicts that target the ribosome. At least three rescue pathways (centered on the prfH/RFH, baeRF-1, and C12orf65 RF-PH domains), were likely innovated in response to such conflicts.


Assuntos
Hidrolases de Éster Carboxílico/genética , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/genética , Ribossomos/genética , Sequência de Aminoácidos , Animais , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Evolução Molecular , Humanos , Modelos Moleculares , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Filogenia , Biossíntese de Proteínas , Domínios Proteicos , Ribossomos/metabolismo
7.
Enzyme Microb Technol ; 126: 41-49, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31000163

RESUMO

The limited thermostability of Yarrowia lipolytica lipase 2 (Lip2) hampers its industrial application. To improve its thermostability, we combined single disulfide bonds which our group identified previously. In this study, combining different regional disulfide bonds had greater effect than combining same regional disulfide bonds. Furthermore, mutants with 4, 5, and 6 disulfide bonds exhibited dramatically enhanced thermostability. Compared with the wild-type, sextuple mutant 6s displayed a 22.53 and 31.23 ℃ increase in the melting temperature (Tm) and the half loss temperature at 15 min (T15 50), respectively, with greater pH stability and a wider reaction pH range. Molecular dynamics simulation revealed that multiple disulfide bonds resulted in more rigid structures of mutants 4s, 5s and 6s, and prolonged enzyme unfolding times. Moreover, secretions of mutants 5s and 6s were significantly increased by 60% and 80% by co-expressing with the chaperone protein disulfide isomerase (PDI), which mitigated the reduced production issue caused by multiple disulfide bonds. Results of this study indicated that enhanced heat endurance giving more potential for industrial application.


Assuntos
Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Dissulfetos/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Temperatura Ambiente , Yarrowia/enzimologia , Hidrolases de Éster Carboxílico/genética , Estabilidade Enzimática , Proteínas Fúngicas/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/metabolismo , Fatores de Transcrição/metabolismo
8.
Chem Biol Interact ; 306: 89-95, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30986387

RESUMO

Human butyrylcholinesterase (BChE) is known as a safe and effective protein for detoxification of organophosphorus (OP) nerve agents. Its rationally designed mutants with considerably improved catalytic activity against cocaine, known as cocaine hydrolases (CocHs), are recognized as the most promising drug candidates for the treatment of cocaine abuse. However, it is a grand challenge to efficiently produce active recombinant BChE and CocHs with a sufficiently long biological half-life. In the present study, starting from a promising CocH, known as CocH3 (i.e. A199S/F227A/S287G/A328W/Y332G mutant of human BChE), which has a ~2000-fold improved catalytic activity against cocaine compared to wild-type BChE, we designed an N-terminal fusion protein, Fc(M3)-(PAPAP)2-CocH3, which was constructed by fusing Fc of human IgG1 to the N-terminal of CocH3 and further optimized by inserting a linker between the two protein domains. Without lowering the enzyme activity, Fc(M3)-(PAPAP)2-CocH3 expressed in Chinese hamster ovary (CHO) cells has not only a long biological half-life of 105 ±â€¯7 h in rats, but also a high yield of protein expression. Particularly, Fc(M3)-(PAPAP)2-CocH3 has a ~21-fold increased protein expression yield in CHO cells compared to CocH3 under the same experimental conditions. Given the observations that Fc(M3)-(PAPAP)2-CocH3 has not only a high catalytic activity against cocaine and a long biological half-life, but also a high yield of protein expression, this new protein entity reported in this study would be a more promising candidate for therapeutic treatment of cocaine overdose and addiction.


Assuntos
Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/química , Fragmentos Fc das Imunoglobulinas/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Animais , Células CHO , Hidrolases de Éster Carboxílico/genética , Cricetulus , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética
9.
Microb Cell Fact ; 18(1): 44, 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30841891

RESUMO

BACKGROUND: Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. RESULTS: We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. CONCLUSION: We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development.


Assuntos
Proteínas de Bactérias/genética , Hidrolases de Éster Carboxílico/genética , Proteínas de Membrana/genética , Mycobacterium marinum/genética , Ovalbumina/metabolismo , Fatores de Virulência/genética , Antígenos de Bactérias/genética , Proteínas de Transporte/genética , Mutagênese , Mutação , Ovalbumina/genética , Sistemas de Secreção Tipo VII/genética , Sistemas de Secreção Tipo VII/metabolismo
10.
Int J Mol Sci ; 20(6)2019 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-30884842

RESUMO

Photosynthetic properties and transcriptomic profiles of green and white sectors of Ficus microcarpa (c.v. milky stripe fig) leaves were examined in naturally variegated plants. An anatomic analysis indicated that chloroplasts of the white sectors contained a higher abundance of starch granules and lacked stacked thylakoids. Moreover, no photosynthetic rate was detected in the white sectors. Transcriptome profile and differential expressed gene (DEG) analysis showed that genes encoding PSII core proteins were down-regulated in the white sectors. In genes related to chlorophyll metabolism, no DEGs were identified in the biosynthesis pathway of chlorophyll. However, genes encoding the first step of chlorophyll breakdown were up-regulated. The repression of genes involved in N-assimilation suggests that the white sectors were deprived of N. The mutation in the transcription factor mitochondrial transcription termination factor (mTERF) suggests that it induces colorlessness in leaves of the milky stripe fig.


Assuntos
Ficus/genética , Fotossíntese/genética , Proteínas de Plantas/genética , Transcriptoma/genética , Arabidopsis/genética , Hidrolases de Éster Carboxílico/genética , Clorofila/genética , Cloroplastos/genética , Ficus/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteólise , Tilacoides/genética
11.
Photochem Photobiol Sci ; 18(5): 1280-1289, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30907896

RESUMO

Softening processes after ripening are a major factor contributing to the perishability of fleshy fruit and, together with mechanical damage, represent the onset of physiological decay. Softening involves multiple co-ordinated events leading to modifications of the cell wall architecture. Several studies described that UV-B radiation positively affects both the nutraceutical and aesthetical qualities of fruit. However, very few studies investigated the effect of UV-B irradiation on the activity of cell wall-related enzymes. This research aimed at studying how different UV-B treatments (10 min and 60 min) affect the activity of cell wall-modifying enzymes (pectin methylesterase, polygalacturonase and ß-galactosidase) together with the expression of some of their isoforms up to 36 h after UV-B treatment of peach (cv. Fairtime, melting phenotype) fruit. Results revealed that UV-B radiation did not affect the soluble solid content and the titratable acidity, two important parameters influencing consumers' choice and taste. In contrast, UV-B was effective at reducing the loss of firmness 24 h after the 60 min irradiation. Generally, a lower activity of the hydrolytic enzymes compared to untreated fruit was observed, regardless of the UV-B dose. However, gene expression did not reflect the corresponding enzymatic activity. Based on these results, UV-B irradiation might be a successful tool in reducing the loss of firmness of peach fruit during post-harvest, thus improving their quality and shelf-life.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/enzimologia , Frutas/metabolismo , Poligalacturonase/metabolismo , Prunus persica/metabolismo , beta-Galactosidase/metabolismo , Hidrolases de Éster Carboxílico/genética , Frutas/genética , Oxirredução , Fenótipo , Poligalacturonase/genética , Prunus persica/genética , RNA/genética , RNA/isolamento & purificação , RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Raios Ultravioleta , beta-Galactosidase/genética
12.
Int J Pediatr Otorhinolaryngol ; 121: 143-149, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30909120

RESUMO

OBJECTIVES: Although hearing loss is a well-known symptom of mitochondria-related disorders, it is not clear how often it is a congenital and cochlear impairment. The Newborn Hearing Screening Program (NHSP) enables to distinguish congenital cochlear deafness from an acquired hearing deficit. The initial aim of the study was to research the frequency of the congenital cochlear hearing loss among patients with various gene defects resulting in mitochondrial disorders. The research process brought on an additional gain: basing on our preliminary study group of 80 patients, in 12 patients altogether we identified two defected genes responsible for mitochondrial disorders, whose carriers did not pass the NHSP. Finally, these patients were diagnosed with the congenital cochlear deafness. MATERIAL AND METHODS: The results of the NHSP in the patients with mitochondrial disorders diagnosed in our tertiary reference center were analyzed. Only the cases with confirmed mutations were qualified for the study group. The NHSP database included 80 patients with mutations in 31 different genes: 25 nuclear-encoded and 6 mtDNA-encoded. We searched the literature for the presence of a congenital hearing impairment (CHI) in mitochondrial disorders caused by changes in 278 already known genes. RESULTS: For 68 patients from the study group the NHSP test indicated a proper cochlear function and thus suggested normal hearing. For 12 mitochondrial patients, the NHSP test indicated the requirement for the further audiological diagnosis, and finally CHI was confirmed in 8 of them. This latter subset included patients with pathogenic variants in RRM2B and SERAC1, known as "deafness-causing genes". Contrary to our initial expectations, the patients carrying mutations in other "deafness-causing genes": MPV17, POLG, COX10, as well as other mitochondria-related genes, all reported in literature, did not indicate any CHI following the NHSP test. CONCLUSION: Our study indicates that the cochlear CHI is a phenotypic feature of the RRM2B and SERAC1 related defects. The diagnosis of the CHI following the NHSP allows to early distinguish those defects from other mitochondria-related disorders in which the NHSP test result is correct. Wider studies are needed to assess the significance of this observation.


Assuntos
Hidrolases de Éster Carboxílico/genética , Proteínas de Ciclo Celular/genética , Surdez/genética , Perda Auditiva Neurossensorial/genética , Doenças Mitocondriais/genética , Ribonucleotídeo Redutases/genética , Adolescente , Criança , Pré-Escolar , DNA Mitocondrial , Surdez/congênito , Feminino , Perda Auditiva Neurossensorial/congênito , Testes Auditivos , Heterozigoto , Hospitais , Humanos , Lactente , Recém-Nascido , Masculino , Doenças Mitocondriais/complicações , Doenças Mitocondriais/diagnóstico , Mutação , Triagem Neonatal , Polônia
13.
Appl Microbiol Biotechnol ; 103(9): 3863-3874, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30863878

RESUMO

Four cutinase genes are encoded in the genome of the saprophytic fungus Aspergillus nidulans, but only two of them have proven to codify for active cutinases. However, their overall roles in cutin degradation are unknown, and there is scarce information on the regulatory effectors of their expression. In this work, the expression of the cutinase genes was assayed by multiplex qRT-PCR in cultures grown in media containing both inducer and repressor carbon sources. The genes ancut1 and ancut2 were induced by cutin and its monomers, while ancut3 was constitutively expressed. Besides, cutin induced ancut4 only under oxidative stress conditions. An in silico analysis of the upstream regulatory sequences suggested binding regions for the lipid metabolism transcription factors (TF) FarA for ancut1 and ancut2 while FarB for ancut3. For ancut4, the analysis suggested binding to NapA (the stress response TF). These binding possibilities were experimentally tested by transcriptional analysis using the A. nidulans mutants ANΔfarA, ANΔfarB, and ANΔnapA. Regarding cutin degradation, spectroscopic and chromatographic methods showed similar products from ANCUT1 and ANCUT3. In addition, ANCUT1 produced 9,10-dihydroxy hexadecanoic acid, suggesting an endo-cleavage action of this enzyme. Regarding ANCUT2 and ANCUT4, they produced omega fatty acids. Our results confirmed the cutinolytic activity of the four cutinases, allowed identification of their specific roles in the cutinolytic system and highlighted their differences in the regulatory mechanisms and affinity towards natural substrates. This information is expected to impact the cutinase production processes and broaden their current biotechnological applications.


Assuntos
Aspergillus nidulans/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Lipídeos de Membrana/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Hidrolases de Éster Carboxílico/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
J Biochem ; 166(2): 149-156, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30825308

RESUMO

A cutinase from Saccharomonospora viridis AHK190, Cut190, can hydrolyze polyethylene terephthalate and has a unique feature that the activity and stability are regulated by Ca2+ binding. Our recent structural and functional analyses showed three Ca2+ binding sites and their respective roles. Here, we analysed the binding thermodynamics of Mn2+, Zn2+ and Mg2+ to Cut190 and their effects on the catalytic activity and thermal stability. The binding affinities of Mn2+ and Zn2+ were higher than that of Mg2+ and are all entropy driven with a binding stoichiometry of three, one and one for Zn2+, Mn2+ and Mg2+, respectively. The catalytic activity was measured in the presence of the respective metals, where the activity of 0.25 mM Mn2+ was comparable to that of 2.5 mM Ca2+. Our 3D Reference Interaction Site Model calculations suggested that all the ions exhibited a high occupancy rate for Site 2. Thus, Mn2+ and Mg2+ would most likely bind to Site 2 (contributes to stability) with high affinity, while to Sites 1 and 3 (contributes to activity) with low affinity. We elucidate the metal-dependent structural and functional properties of Cut190 and show the subtle balance on structure stability and flexibility is controlled by specific metal ions.


Assuntos
Actinobacteria/enzimologia , Cálcio/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Zinco/metabolismo , Sítios de Ligação , Cálcio/química , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Estabilidade Enzimática , Magnésio/química , Manganês/química , Modelos Moleculares , Mutação , Termodinâmica , Zinco/química
15.
Plant Cell Physiol ; 60(6): 1250-1259, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30796452

RESUMO

Degradation of the storage compound triacylglycerol (TAG) is a crucial process in response to environmental stimuli. In microalgae, this process is important for re-growth when conditions become favorable after cells have experienced stresses. Mobilization of TAG is initiated by actions of lipases causing the release of glycerol and free fatty acids, which can be further broken down for energy production or recycled to synthesize membrane lipids. Although key enzymes in the process, TAG lipases remain to be characterized in the model green alga Chlamydomonas reinhardtii. Here, we describe the functional analysis of a putative TAG lipase, i.e. LIP4, which shares 44% amino acid identity with the major TAG lipase in Arabidopsis (SUGAR DEPENDENT1-SDP1). The LIP4 transcript level was downregulated during nitrogen deprivation when TAG accumulates, but was upregulated during nitrogen resupply (NR) when TAG was degraded. Both artificial microRNA and insertional mutants showed a delay in TAG mobilization during NR. The difference in TAG degradation was more pronounced when the cultures were incubated without acetate in the dark. Furthermore, the lip4 insertional mutants over-accumulated TAG during optimal growth conditions. Taken together, the results suggest to us that LIP4 likely acts as a TAG lipase and plays a role in TAG homeostasis in Chlamydomonas.


Assuntos
Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Lipase/metabolismo , Triglicerídeos/metabolismo , Proteínas de Algas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Hidrolases de Éster Carboxílico/genética , Chlamydomonas reinhardtii/genética , Lipase/genética , Filogenia
16.
Microb Cell Fact ; 18(1): 40, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808422

RESUMO

BACKGROUND: Due to various environmental problems, biodegradable polymers such as poly (3-hydroxybutyrate) (PHB) have gained much attention in recent years. Purple non-sulfur (PNS) bacteria have various attractive characteristics useful for environmentally harmless PHB production. However, production of PHB by PNS bacteria using genetic engineering has never been reported. This study is the first report of a genetically engineered PNS bacterial strain with a high PHB production. RESULTS: We constructed a poly (3-hydroxyalkanoate) depolymerase (phaZ) gene-disrupted Rhodobacter sphaeroides HJ strain. This R. sphaeroides HJΔphaZ (pLP-1.2) strain showed about 2.9-fold higher volumetric PHB production than that of the parent HJ (pLP-1.2) strain after 5 days of culture. The HJΔphaZ strain was further improved for PHB production by constructing strains overexpressing each of the eight genes including those newly found and annotated as PHB biosynthesis genes in the KEGG GENES Database. Among these constructed strains, all of gene products exhibited annotated enzyme activities in the recombinant strain cells, and HJΔphaZ (phaA3), HJΔphaZ (phaB2), and HJΔphaZ (phaC1) showed about 1.1-, 1.1-, and 1.2-fold higher volumetric PHB production than that of the parent HJΔphaZ (pLP-1.2) strain. Furthermore, we constructed a strain that simultaneously overexpresses all three phaA3, phaB2, and phaC1 genes; this HJΔphaZ (phaA3/phaB2/phaC1) strain showed about 1.7- to 3.9-fold higher volumetric PHB production (without ammonium sulfate; 1.88 ± 0.08 g l-1 and with 100 mM ammonium sulfate; 0.99 ± 0.05 g l-1) than those of the parent HJ (pLP-1.2) strain grown under nitrogen limited and rich conditions, respectively. CONCLUSION: In this study, we identified eight different genes involved in PHB biosynthesis in the genome of R. sphaeroides 2.4.1, and revealed that their overexpression increased PHB accumulation in an R. sphaeroides HJ strain. In addition, we demonstrated the effectiveness of a phaZ disruption for high PHB accumulation, especially under nitrogen rich conditions. Furthermore, we showed that PNS bacteria may have some unidentified genes involved in poly (3-hydroxyalkanoates) (PHA) biosynthesis. Our findings could lead to further improvement of environmentally harmless PHA production techniques using PNS bacteria.


Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Nitrogênio/metabolismo , Rhodobacter sphaeroides/genética , Proteínas de Bactérias/genética , Hidrolases de Éster Carboxílico/genética , Engenharia Genética , Polímeros , Rhodobacter sphaeroides/metabolismo
17.
Proc Natl Acad Sci U S A ; 116(9): 3764-3773, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30755523

RESUMO

Commensal and pathogenic bacteria hydrolyze host lipid substrates with secreted lipases and phospholipases for nutrient acquisition, colonization, and infection. Bacterial lipase activity on mammalian lipids and phospholipids can promote release of free fatty acids from lipid stores, detoxify antimicrobial lipids, and facilitate membrane dissolution. The gram-positive bacterium Staphylococcus aureus secretes at least two lipases, Sal1 and glycerol ester hydrolase (Geh), with specificities for short- and long-chain fatty acids, respectively, each with roles in the hydrolysis of environmental lipids. In a recent study from our group, we made the unexpected observation that Geh released by S. aureus inhibits activation of innate immune cells. Herein, we investigated the possibility that S. aureus lipases interface with the host immune system to blunt innate immune recognition of the microbe. We found that the Geh lipase, but not other S. aureus lipases, prevents activation of innate cells in culture. Mutation of geh leads to enhancement of proinflammatory cytokine production during infection, increased innate immune activity, and improved clearance of the bacterium in infected tissue. These in vitro and in vivo effects on innate immunity were not due to direct functions of the lipase on mammalian cells, but rather a result of inactivation of S. aureus lipoproteins, a major pathogen-associated molecular pattern (PAMP) of extracellular gram-positive bacteria, via ester hydrolysis. Altogether, these studies provide insight into an adaptive trait that masks microbial recognition by innate immune cells through targeted inactivation of a broadly conserved PAMP.


Assuntos
Hidrolases de Éster Carboxílico/genética , Imunidade Inata/genética , Lipase/genética , Infecções Estafilocócicas/enzimologia , Staphylococcus aureus/enzimologia , Animais , Hidrolases de Éster Carboxílico/imunologia , Interações Hospedeiro-Patógeno/genética , Ligantes , Lipase/imunologia , Lipólise/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Mutação , Pele/enzimologia , Pele/metabolismo , Pele/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade
18.
Mol Plant Microbe Interact ; 32(6): 673-684, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30598046

RESUMO

Mitogen-activated protein kinase (MAPK) cascades serve as unified signaling modules in plant development and defense response. Previous reports demonstrated an essential role of Arabidopsis GLIP1, a member of the GDSL-like-motif lipase family, in both local and systemic resistance. GLIP1 expression is highly induced by pathogen attack. However, the one or more signaling pathways involved are unknown. Here, we report that two pathogen-responsive MAPKs, MPK3 and MPK6, are implicated in regulating gene expression of GLIP1 as well as GLIP3 and GLIP4. After gain-of-function activation, MPK3 and MPK6 can strongly induce the expression of GLIP1, GLIP3, and GLIP4. Both GLIP1 and GLIP3 contribute to the plant resistance to Botrytis cinerea. WRKY33, a MPK3/MPK6 substrate, is essential for the MPK3/MPK6-dependent GLIP1 induction. In addition, WRKY2 and WRKY34, two close homologs of WRKY33, have a minor effect in MPK3/MPK6-regulated GLIP1 expression in B. cinerea-infected plants. Chromatin immunoprecipitation-quantitative polymerase chain reaction analysis demonstrated that the GLIP1 gene is a direct target of WRKY33. In addition, we demonstrated that MPK3/MPK6-induced GLIP1 expression is independent of ethylene and jasmonic acid, two important hormones in plant defense. Our results provide insights into the regulation of the GLIP family at the transcriptional level in plant immunity.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Hidrolases de Éster Carboxílico , Resistência à Doença , Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno , Fatores de Transcrição , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Botrytis/fisiologia , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/imunologia , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas/imunologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , Fatores de Transcrição/imunologia
19.
N Biotechnol ; 51: 14-20, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-30685332

RESUMO

The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants.


Assuntos
Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Evolução Molecular Direcionada , Fusarium/enzimologia , Simulação de Acoplamento Molecular , Hidrolases de Éster Carboxílico/metabolismo , Reação em Cadeia da Polimerase
20.
Malar J ; 18(1): 22, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683097

RESUMO

BACKGROUND: Since malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium, a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. PfGluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites. METHODS: Plasmodium vivax glucose 6-phosphate dehydrogenase (PvG6PD) was cloned, recombinantly produced in Escherichia coli, purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium. RESULTS: Here the recombinant production and characterization of PvG6PD, the C-terminal and NADPH-producing part of PvGluPho, is described. A comparison with PfG6PD (the NADPH-producing part of PfGluPho) indicates that the P. vivax enzyme has higher KM values for the substrate and cofactor. Like the P. falciparum enzyme, PvG6PD is hardly affected by S-glutathionylation and moderately by S-nitrosation. Since there are several naturally occurring variants of PfGluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on PfGluPho. ML304, a recently developed PfGluPho inhibitor, was verified to also be active on PvG6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity. CONCLUSION: The characterization of PvG6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring PfGluPho variants studied here are unlikely to have a major impact on the parasites' redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up.


Assuntos
Hidrolases de Éster Carboxílico/genética , Glucosefosfato Desidrogenase/genética , Malária Vivax/genética , Complexos Multienzimáticos/genética , Proteínas de Protozoários/genética , Hidrolases de Éster Carboxílico/metabolismo , Clonagem Molecular , Citosol/metabolismo , Escherichia coli/metabolismo , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/metabolismo , Cinética , Malária Vivax/enzimologia , Malária Vivax/metabolismo , Complexos Multienzimáticos/metabolismo , Oxirredução , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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