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1.
J Enzyme Inhib Med Chem ; 35(1): 377-382, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31856608

RESUMO

The inhibition of δ- and η-class carbonic anhydrases (CAs; EC 4.2.1.1) was poorly investigated so far. Only one δ-CA, TweCA from the diatom Thalassiosira weissflogii, and one η-CA, PfCA, from Plasmodium falciparum, have been cloned and characterised to date. To enrich δ- and η-CAs inhibition profiles, a panel of 22 phenols was investigated for TweCA and PfCA inhibition. Some derivatives showed effective, sub-micromolar inhibition of TweCA (KIs 0.81-65.4 µM) and PfCA (KIs 0.62-78.7 µM). A subset of compounds demonstrated a significant selectivity for the target CAs over the human physiologically relevant ones. This study promotes the identification of new potent and selective inhibitors of TweCA and PfCA, which could be considered as leads for finding molecular probes in the study of carbon fixation processes (in which TweCA and orthologue enzymes are involved) or drug candidates in the treatment of malaria.


Assuntos
Antiprotozoários/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Diatomáceas/enzimologia , Fenóis/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Antiprotozoários/síntese química , Antiprotozoários/química , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Fenóis/síntese química , Fenóis/química , Plasmodium falciparum/enzimologia , Relação Estrutura-Atividade
2.
J Biosci Bioeng ; 128(6): 755-765, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31277909

RESUMO

The diatom microalgal Phaeodactylum tricornutum accumulates a large amount of fucoxanthin. Carotenoids hydroxylases (CHYs) play key roles in fucoxanthin biosynthesis in diatoms. However, not any type of CHYs had been identified in P. tricornutum. In this study, two genes (designated Ptrcyp97b1 and Ptrcyp97b2) were cloned, identified and functionally characterized. They shared high sequence identity (50-94 %) with lutein deficient 1-like proteins from other eukaryotes. The typical catalytic active motifs of cytochrome P450s (CYP) were detected in the amino acid sequences of PtrCYP97B1 and PtrCYP97B2. The two genes were probably due to gene duplication. Ptrcyp97b1 and Ptrcyp97b2 transcriptional expression was up-regulated with distinct patterns under high light conditions. The metabolic profiles of the major carotenoids (ß-carotene, zeaxanthin, diadinoxanthin, diatoxanthin and fucoxanthin) were determined based on the high performance liquid chromatography method. The fucoxanthin and diatoxanthin contents were increased, while the ß-carotene content was decreased. By truncation of the N-terminal trans-membrane anchor or chloroplast transit peptide and addition of a 6 × His-tag, PtrCYP97B1 and PtrCYP97B2 were separately heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. Functional analysis showed that PrtCYP97B2 was able to catalyze the hydroxylation of the ß-rings of ß-carotene to produce zeaxanthin in ß-carotene-accumulating E. coli BL21(DE3) cells. PtrCYP97B1 might have the ability to catalyze the hydroxylation of other substrates other than ß-carotene. These results contribute to the further elucidation of xanthophyll biosynthesis in diatoms.


Assuntos
Carotenoides/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Diatomáceas/enzimologia , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Diatomáceas/genética , Escherichia coli/genética , Hidroxilação
3.
J Enzyme Inhib Med Chem ; 34(1): 644-650, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30727781

RESUMO

Famotidine, an antiulcer drug belonging to the H2 antagonists class of pharmacological agents, was recently shown to potently inhibit human (h) and bacterial carbonic anhydrases (CAs, EC 4.2.1.1). We investigated the inhibitory effects of famotidine against all classes of CAs from the pathogenic bacteria Vibrio cholerae, Burkholderia pseudomallei and Mycobacterium tuberculosis Rv3273 ß-CA, as well as the CAs from the nonpathogenic bacteria/cyanobacteria Sulfurihydrogenibium yellowstonensis, S. azorense, Pseudoalteromonas haloplanktis, Colwellia psychrerythraea and Nostoc commune. The δ- and ζ-CAs from the diatom Thalassiosira weissflogii, the fungal enzymes from Cryptococcus neoformans, Candida glabrata and Malassezia globosa, as well as the protozoan enzymes from Trypanosoma cruzi and Plasmodium falciparum, were also investigated. Anopheles gambiae ß-CA was also investigated. All these enzymes were effectively inhibited by famotidine, with affinities between the low nanomolar to the micromolar range. The best inhibition was observed against C. glabrata ß-CA and TweCAζ, with KIs ranging between 13.6 and 22.1 nM.


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Famotidina/farmacologia , Bactérias/enzimologia , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Clorófitas/enzimologia , Diatomáceas/enzimologia , Famotidina/síntese química , Famotidina/química , Fungos/enzimologia , Humanos , Estrutura Molecular , Plasmodium falciparum/enzimologia , Trypanosoma cruzi/enzimologia
4.
J Enzyme Inhib Med Chem ; 34(1): 510-518, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30688123

RESUMO

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which started to be investigated in detail in pathogenic, as well as non-pathogenic species since their pivotal role is to accelerate the physiological CO2 hydration/dehydration reaction significantly. Here, we propose the marine unicellular diatom Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of CA inhibitors (CAIs). Seven inhibitors belonging to the sulphonamide type and possessing a diverse scaffold have been explored for their in vitro inhibition of the whole diatom CAs and the in vivo inhibitory effect on the growth of P. tricornutum. Interesting, inhibition of growth was observed, in vivo, demonstrating that this diatom is a good model for testing the cell wall penetrability of this class of pharmacological agents. Considering that many pathogens are difficult and dangerous to grow in the laboratory, the growth inhibition of P. tricornutum with different such CAIs may be subsequently used to design inhibition studies of CAs from pathogenic organisms.


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Diatomáceas/efeitos dos fármacos , Sulfonamidas/farmacologia , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/isolamento & purificação , Permeabilidade da Membrana Celular/efeitos dos fármacos , Diatomáceas/enzimologia , Diatomáceas/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química
5.
Nat Microbiol ; 4(2): 226-233, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30478288

RESUMO

Steroids are essential triterpenoid molecules that are present in all eukaryotes and modulate the fluidity and flexibility of cell membranes. Steroids also serve as signalling molecules that are crucial for growth, development and differentiation of multicellular organisms1-3. The steroid biosynthetic pathway is highly conserved and is key in eukaryote evolution4-7. The flavoprotein squalene epoxidase (SQE) catalyses the first oxygenation reaction in this pathway and is rate limiting. However, despite its conservation in animals, plants and fungi, several phylogenetically widely distributed eukaryote genomes lack an SQE-encoding gene7,8. Here, we discovered and characterized an alternative SQE (AltSQE) belonging to the fatty acid hydroxylase superfamily. AltSQE was identified through screening of a gene library of the diatom Phaeodactylum tricornutum in a SQE-deficient yeast. In accordance with its divergent protein structure and need for cofactors, we found that AltSQE is insensitive to the conventional SQE inhibitor terbinafine. AltSQE is present in many eukaryotic lineages but is mutually exclusive with SQE and shows a patchy distribution within monophyletic clades. Our discovery provides an alternative element for the conserved steroid biosynthesis pathway, raises questions about eukaryote metabolic evolution and opens routes to develop selective SQE inhibitors to control hazardous organisms.


Assuntos
Eucariotos/enzimologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Esteroides/biossíntese , Vias Biossintéticas , Coenzimas , Diatomáceas/enzimologia , Diatomáceas/genética , Diatomáceas/metabolismo , Eucariotos/classificação , Eucariotos/genética , Eucariotos/metabolismo , Expressão Gênica , Teste de Complementação Genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/química , Filogenia , Conformação Proteica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esqualeno/análogos & derivados , Esqualeno/metabolismo , Esqualeno Mono-Oxigenase/química , Esqualeno Mono-Oxigenase/genética , Esqualeno Mono-Oxigenase/metabolismo , Terbinafina/farmacologia
6.
Ecotoxicol Environ Saf ; 169: 654-661, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30496998

RESUMO

The environmental risk issues of p-choroaniline have been concerned by the widespread application and transportation of this important chemical intermediate. The information about the toxicity of p-chloroaniline was mainly concentrated on freshwater organisms while the current knowledge on marine organisms was scarce yet. In this study, acute toxicity and toxic physiology characteristic of p-chloroaniline to Phaeodactylum tricornutum (P. tricornutum) were first determined. In the acute experiments, the effect of the p-choroaniline to P. tricornutum showed time- and dose-dependent response, which the half maximum effective concentration (EC50) at 24 h, 48 h and 96 h was 35.35, 20.10 and 10.00 mgL-1, respectively. Toxic physiology assays in P. tricornutum indicated that the p-choroaniline induced significant changes of photosynthetic pigments (Chl-a, Chl-b, Caro, Chl-a/b and Chl-(a+b)/Caro), Chlorophyll fluorescence parameters (Fv/Fm, ETR, qP and NPQ), rates of photosynthetic O2 release and respiration O2 consumption, and antioxidant enzyme activities (SOD, CAT). The obvious decrease of Fv/Fm, ETR and chl-a in low p-choroaniline treatments (≤ 5.00 mgL-1) compared with the control could be observed, which implied that these parameters could be taken as sensitive indicators for the environmental assessment. Meanwhile, the activities of SOD and CAT significant increase in p-choroaniline stress after 24 h and the extent of the increase has fallen after 96 h. These toxicity data obtained here might provide available basic data for the ecological risk assessment of p-choroaniline pollution.


Assuntos
Compostos de Anilina/toxicidade , Antioxidantes/metabolismo , Diatomáceas , Consumo de Oxigênio/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Clorofila/metabolismo , Diatomáceas/efeitos dos fármacos , Diatomáceas/enzimologia , Diatomáceas/crescimento & desenvolvimento , Testes de Toxicidade Aguda , Testes de Toxicidade Subcrônica
7.
New Phytol ; 221(3): 1303-1316, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30216452

RESUMO

Diatom dominance in contemporary aquatic environments indicates that they have developed unique and effective mechanisms to cope with the rapid and considerable fluctuations that characterize these environments. In view of their evolutionary history from a secondary endosymbiosis, inter-organellar regulation of biochemical activities may be of particular relevance. Diatom mitochondrial alternative oxidase (AOX) is believed to play a significant role in supplying chloroplasts with ATP produced in the mitochondria. Using the model diatom Phaeodactylum tricornutum we generated AOX knockdown lines, and followed sensitivity to stressors, photosynthesis and transcriptome and metabolome profiles of wild-type and knockdown lines. We show here that expression of the AOX gene is upregulated by various stresses including H2 O2 , heat, high light illumination, and iron or nitrogen limitation. AOX knockdown results in hypersensitivity to stress. Knockdown lines also show significantly reduced photosynthetic rates and their chloroplasts are more oxidized. Comparisons of transcriptome and metabolome profiles suggest a strong impact of AOX activity on gene expression, which is carried through to the level of the metabolome. Our data provide evidence for the involvement of mitochondrial AOX in processes central to the cell biology of diatoms, revealing that cross-talk between mitochondria and chloroplasts is crucial for maintaining sensitivity to changing environments.


Assuntos
Organismos Aquáticos/enzimologia , Cloroplastos/metabolismo , Diatomáceas/enzimologia , Diatomáceas/fisiologia , Regulação para Baixo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Antioxidantes/metabolismo , Organismos Aquáticos/fisiologia , Glutationa/metabolismo , Metabolômica , Oxirredução , Fotossíntese , Transcriptoma/genética
8.
Environ Microbiol ; 20(11): 4157-4169, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30246477

RESUMO

Dimethylsulfide (DMS), a dominant organic sulfur species in the surface ocean, may act as a signalling molecule and contribute to mutualistic interactions between bacteria and marine algae. These proposed functions depend on the DMS concentration in the vicinity of microorganisms. Here, we modelled the DMS enrichment at the surface of DMS-releasing marine algal cells as a function of DMS production rate, algal cell radius and turbulence. Our results show that the DMS concentration at the surface of unstressed phytoplankton with low DMS production rates can be enriched by <1 nM, whereas for mechanically stressed algae with high activities of the enzyme DMSP-lyase (a coccolithophore and a dinoflagellate) DMS cell surface enrichments can reach ~10 nM, and could potentially reach µM levels in large cells. These DMS enrichments are much higher than the median DMS concentration in the surface ocean (1.9 nM), and thus may attract and support the growth of bacteria living in the phycosphere. The bacteria in turn may provide photoactive iron chelators (siderophores) that enhance algal iron uptake and provide algal growth factors such as auxins and vitamins. The present study highlights new insights on the extent and impact of microscale DMS enrichments at algal surfaces, thereby contributing to our understanding of the potential chemoattractant and mutualistic roles of DMS in marine microorganisms.


Assuntos
Haptófitas/metabolismo , Fitoplâncton/metabolismo , Sulfetos/metabolismo , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Diatomáceas/enzimologia , Diatomáceas/genética , Diatomáceas/metabolismo , Dinoflagelados/enzimologia , Dinoflagelados/genética , Dinoflagelados/metabolismo , Ecossistema , Haptófitas/enzimologia , Haptófitas/genética , Ferro/metabolismo , Fitoplâncton/enzimologia , Fitoplâncton/genética , Água do Mar/microbiologia , Água do Mar/parasitologia , Sideróforos/metabolismo , Sulfetos/análise
9.
J Enzyme Inhib Med Chem ; 33(1): 1194-1198, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30044657

RESUMO

The inhibition of α-, ß-, γ-, and δ-class carbonic anhydrases (CAs, EC 4.2.1.1) from bacteria (Vibrio cholerae and Porphyromonas gingivalis) and diatoms (Thalassiosira weissflogii) with a panel of N'-aryl-N-hydroxy-ureas is reported. The α-/ß-CAs from V. cholerae (VchCAα and VchCAß) were effectively inhibited by some of these derivatives, with KIs in the range of 97.5 nM - 7.26 µM and 52.5 nM - 1.81 µM, respectively, whereas the γ-class enzyme VchCAγ was less sensitive to inhibition (KIs of 4.75 - 8.87 µM). The ß-CA from the pathogenic bacterium Porphyromonas gingivalis (PgiCAß) was not inhibited by these compounds (KIs > 10 µM) whereas the corresponding γ-class enzyme (PgiCAγ) was effectively inhibited (KIs of 59.8 nM - 6.42 µM). The δ-CA from the diatom Thalassiosira weissflogii (TweCAδ) showed effective inhibition with these derivatives (KIs of 33.3 nM - 8.74 µM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (KIs > 10 µM), this class of derivatives may lead to the development of CA inhibitors selective for bacterial/diatom enzymes over their human counterparts and thus to anti-infectives or agents with environmental applications.


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Diatomáceas/enzimologia , Hidroxiureia/farmacologia , Isoenzimas/antagonistas & inibidores , Porphyromonas gingivalis/enzimologia , Vibrio cholerae/enzimologia , Inibidores da Anidrase Carbônica/isolamento & purificação , Humanos , Hidroxiureia/química
10.
Genome Biol Evol ; 10(9): 2310-2325, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30060189

RESUMO

The establishment of the mitochondrion is seen as a transformational step in the origin of eukaryotes. With the mitochondrion came bioenergetic freedom to explore novel evolutionary space leading to the eukaryotic radiation known today. The tight integration of the bacterial endosymbiont with its archaeal host was accompanied by a massive endosymbiotic gene transfer resulting in a small mitochondrial genome which is just a ghost of the original incoming bacterial genome. This endosymbiotic gene transfer resulted in the loss of many genes, both from the bacterial symbiont as well the archaeal host. Loss of genes encoding redundant functions resulted in a replacement of the bulk of the host's metabolism for those originating from the endosymbiont. Glycolysis is one such metabolic pathway in which the original archaeal enzymes have been replaced by bacterial enzymes from the endosymbiont. Glycolysis is a major catabolic pathway that provides cellular energy from the breakdown of glucose. The glycolytic pathway of eukaryotes appears to be bacterial in origin, and in well-studied model eukaryotes it takes place in the cytosol. In contrast, here we demonstrate that the latter stages of glycolysis take place in the mitochondria of stramenopiles, a diverse and ecologically important lineage of eukaryotes. Although our work is based on a limited sample of stramenopiles, it leaves open the possibility that the mitochondrial targeting of glycolytic enzymes in stramenopiles might represent the ancestral state for eukaryotes.


Assuntos
Blastocystis/metabolismo , Diatomáceas/metabolismo , Glicólise , Mitocôndrias/metabolismo , Evolução Biológica , Blastocystis/citologia , Blastocystis/enzimologia , Blastocystis/genética , Diatomáceas/citologia , Diatomáceas/enzimologia , Diatomáceas/genética , Metabolismo Energético , Genoma Mitocondrial , Mitocôndrias/genética , Simbiose , Transformação Genética
11.
Bioorg Chem ; 80: 261-265, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29966872

RESUMO

The activation of the ζ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii (TweCAζ) incorporating both Zn(II) and Cd(II) at the active site, was investigated for the first time, using a panel of natural and non-natural amino acids and amines. CdTweCAζ was completely insensitive to activation, whereas all these compounds were effective activators of the zinc-containing enzyme ZnTweCAζ, with activation constants ranging between 92 nM and 37.9 µM. The most effective ZnTweCAζ activators were l-adrenaline, 1-(2-aminoethyl)-piperazine and 4-(2-aminoethyl)-morpholine, with KAs in the range of 92-150 nM. l-His, l- and d-Tyr and some pyridyl-alkylamines, had KAs in the range of 0.62-0.98 µM, whereas l-/d-DOPA, d-Trp, histamine, serotonin and l-Asn were the next most efficient activators, with KAs in the range of 1.27-3.19 µM. The least effective activators were l-Phe (KA of 15.4 µM) and l-Asp (KA of 37.9 µM). This in vitro study may be useful for a more complete understanding of the activation processes of various CA enzyme families, of which the ζ-class was scarcely investigated.


Assuntos
Aminas/metabolismo , Aminoácidos/metabolismo , Anidrases Carbônicas/metabolismo , Diatomáceas/enzimologia , Zinco/química , Aminas/química , Aminas/farmacologia , Aminoácidos/química , Aminoácidos/farmacologia , Cádmio/química , Ativação Enzimática/efeitos dos fármacos , Humanos , Isoenzimas/metabolismo , Cinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
12.
J Biol Chem ; 293(34): 13033-13043, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-29925588

RESUMO

The catalytic performance of the major CO2-assimilating enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), restricts photosynthetic productivity. Natural diversity in the catalytic properties of Rubisco indicates possibilities for improvement. Oceanic phytoplankton contain some of the most efficient Rubisco enzymes, and diatoms in particular are responsible for a significant proportion of total marine primary production as well as being a major source of CO2 sequestration in polar cold waters. Until now, the biochemical properties and three-dimensional structures of Rubisco from diatoms were unknown. Here, diatoms from arctic waters were collected, cultivated, and analyzed for their CO2-fixing capability. We characterized the kinetic properties of five and determined the crystal structures of four Rubiscos selected for their high CO2-fixing efficiency. The DNA sequences of the rbcL and rbcS genes of the selected diatoms were similar, reflecting their close phylogenetic relationship. The Vmax and Km for the oxygenase and carboxylase activities at 25 °C and the specificity factors (Sc/o) at 15, 25, and 35 °C were determined. The Sc/o values were high, approaching those of mono- and dicot plants, thus exhibiting good selectivity for CO2 relative to O2 Structurally, diatom Rubiscos belong to form I C/D, containing small subunits characterized by a short ßA-ßB loop and a C-terminal extension that forms a ß-hairpin structure (ßE-ßF loop). Of note, the diatom Rubiscos featured a number of posttranslational modifications of the large subunit, including 4-hydroxyproline, ß-hydroxyleucine, hydroxylated and nitrosylated cysteine, mono- and dihydroxylated lysine, and trimethylated lysine. Our studies suggest adaptation toward achieving efficient CO2 fixation in arctic diatom Rubiscos.


Assuntos
Dióxido de Carbono/metabolismo , Diatomáceas/enzimologia , Processamento de Proteína Pós-Traducional , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/metabolismo , Cristalografia por Raios X , Hidroxilação , Cinética , Nitrosação , Filogenia , Conformação Proteica , Dobramento de Proteína , Ribulose-Bifosfato Carboxilase/genética , Relação Estrutura-Atividade
13.
Mar Biotechnol (NY) ; 20(5): 566-572, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29931608

RESUMO

Photosynthetic microalgae are of burgeoning interest in the generation of commercial bioproducts. Microalgae accumulate high lipid content under adverse conditions, which in turn compromise their growth and hinder their commercial potential. Hence, it is necessary to engineer microalgae to mitigate elevated lipid accumulation and biomass. In this study, we identified acetyl-CoA carboxylase (ACCase) in oleaginous microalga Phaeodactylum tricornutum (PtACC2) and expressed constitutively in the chloroplast to demonstrate the potential of chloroplast engineering. Molecular characterization of transplastomic microalgae revealed that PtACC2 was integrated, transcribed and expressed successfully, and localized in the chloroplast. Enzymatic activity of ACCase was elevated by 3.3-fold, and the relative neutral lipid content increased substantially by 1.77-fold, and lipid content reached up to 40.8% of dry weight. Accordingly, the number and size of oil bodies markedly increased. Fatty acid profiling showed that the content of monounsaturated fatty acids increased, while polyunsaturated fatty acids decreased. This method provides a valuable genetic engineering toolbox for microalgal bioreactors with industrial significance.


Assuntos
Acetil-CoA Carboxilase/genética , Cloroplastos/genética , Diatomáceas/genética , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Graxos Insaturados/biossíntese , Microalgas/genética , Acetil-CoA Carboxilase/metabolismo , Biomassa , Reatores Biológicos , Cloroplastos/enzimologia , Diatomáceas/classificação , Diatomáceas/enzimologia , Expressão Gênica , Metabolismo dos Lipídeos/genética , Engenharia Metabólica/métodos , Microalgas/classificação , Microalgas/enzimologia , Fotossíntese/genética , Filogenia , Plasmídeos/química , Plasmídeos/metabolismo , Transformação Genética
14.
ACS Chem Biol ; 13(8): 2237-2246, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29874034

RESUMO

Humans have lost the ability to convert urate into the more soluble allantoin with the evolutionary inactivation of three enzymes of the uricolytic pathway. Restoration of this function through enzyme replacement therapy can treat severe hyperuricemia and Lesch-Nyhan disease. Through a genomic exploration of natural gene fusions, we found that plants and diatoms independently evolved a fusion protein (allantoin synthase) complementing two human pseudogenes. The 1.85-Å-resolution crystal structure of allantoin synthase from the diatom Phaeodactylum tricornutum provides a rationale for the domain combinations observed in the metabolic pathway, suggesting that quaternary structure is key to the evolutionary success of protein domain fusions. Polyethylene glycol (PEG) conjugation experiments indicate that a PEG-modified form of the natural fusion protein provides advantages over separate enzymes in terms of activity maintenance and manufacturing of the bioconjugate. These results suggest that the combination of different activities in a single molecular unit can simplify the production and chemical modification of recombinant proteins for multifunctional enzyme therapy.


Assuntos
Alantoína/metabolismo , Diatomáceas/enzimologia , Ligases/metabolismo , Vias Biossintéticas , Cristalografia por Raios X , Diatomáceas/química , Diatomáceas/genética , Diatomáceas/metabolismo , Estabilidade Enzimática , Fusão Gênica , Ligases/química , Ligases/genética , Modelos Moleculares , Polietilenoglicóis/química , Conformação Proteica
15.
J Biosci Bioeng ; 126(2): 258-265, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29628268

RESUMO

Microalgal biofuels are desirable alternatives for traditional liquid fuels, but further improvements of productivity are necessary for microalgal biofuels to be economically feasible. As strategies for improving lipid productivity, repression of lipid degradation has a great potential because the lipid accumulation level is determined by a balance between lipid synthesis and degradation. However, studies of lipid degradation and its primary functioning enzyme triacylglycerol (TAG) lipases in microalgae are currently limited. In this study, we report the comprehensive analysis of TAG lipases in the oleaginous diatom Fistulifera solaris JPCC DA0580. First, we confirmed that TAGs in the lipid accumulation organelles, oil bodies, were degraded when the cells were transferred from the nutrient depleted conditions to nutrient replete condition. Further analysis revealed that, after eicosapentaenoic acid (C20:5n-3)-containing TAGs are were degraded, its molecular skeletons were likely to be recycled to produce glycolipids and phospholipids for chloroplast regeneration and cell growth, respectively. Next, we searched putative TAG lipase genes from the draft genome sequence of F. solaris, and discovered 42 candidates based on the amino acid sequence homology analysis. Subsequent transcriptome analysis revealed that 16 of the 42 lipase genes were up-regulated during lipid degradation. Among the up-regulated lipases, a number of enzymes were predicted to localize in endoplasmic reticulum which is closely associated to the lipid accumulation organelles, oil bodies. Our study provided new insights of lipid degradation in oleaginous microalgae, and putative TAG lipases which could be candidates for metabolic engineering in future study to improve microalgal lipid productivity.


Assuntos
Diatomáceas , Lipase/análise , Lipase/genética , Metabolismo dos Lipídeos/genética , Biocombustíveis , Cloroplastos/metabolismo , Diatomáceas/enzimologia , Diatomáceas/genética , Diatomáceas/metabolismo , Ácido Eicosapentaenoico/metabolismo , Retículo Endoplasmático/metabolismo , Indução Enzimática , Perfilação da Expressão Gênica , Lipase/metabolismo , Engenharia Metabólica , Microalgas/enzimologia , Microalgas/genética , Microalgas/metabolismo , Transcriptoma , Triglicerídeos/metabolismo
16.
Plant Physiol Biochem ; 127: 248-255, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29626705

RESUMO

Betaine (trimethylglycine) is an important compatible solute that accumulates in response to abiotic stresses such as drought and salinity. Biosynthetic pathways of betaine have been extensively studied, but it remains to be clarified on algae. A diatom Thalassiosira pseudonana CCMP1335 is an important component of marine ecosystems. Here we show that the genome sequence of Thalassiosira suggests the presence of two biosynthetic pathways for betaine, via three step methylation of glycine and via two step oxidation of choline. The choline oxidation via choline dehydrogenase was suggested and its sequential characteristics were analyzed. A candidate gene TpORF1 for glycine methylation encodes a protein consisted of 574 amino acids with two putative tandem repeat methyltransferase domains. The TpORF1 was expressed in E. coli, and the purified protein was shown to synthesize betaine via three step methylation of glycine and designated as TpGSDMT. The proteins containing C-terminal half or N-terminal half were expressed in E. coli and exhibited the methyl transferase activities with different substrate specificity for glycine, sarcosine and dimethylglycine. Upregulation of TpGSDMT transcription and betaine levels were observed at high salinity, suggesting the importance of TpGSDMT for salt tolerance in T. pseudonana cells.


Assuntos
Betaína , Colina , Diatomáceas/enzimologia , Genoma , Glicina , Metiltransferases , Betaína/análogos & derivados , Betaína/metabolismo , Colina/genética , Colina/metabolismo , Diatomáceas/genética , Glicina/genética , Glicina/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo
17.
J Enzyme Inhib Med Chem ; 33(1): 680-685, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29536765

RESUMO

The activation of the δ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii (TweCAδ) was investigated using a panel of natural and non-natural amino acids and amines. The most effective activator of TweCAδ was d-Tyr (KA of 51 nM), whereas several other amino acids and amines, such as L-His, L-Trp, d-Trp, dopamine and serotonin were submicromolar activators (KAs from 0.51 to 0.93 µM). The most ineffective activator of TweCAδ was 4-amino-l-Phe (18.9 µM), whereas d-His, l-/d-Phe, l-/d-DOPA, l-Tyr, histamine, some pyridyl-alkylamines, l-adrenaline and aminoethyl-piperazine/morpholine were moderately potent activators (KAs from 1.34 to 8.16 µM). For any δ-CA, there are no data on the crystal structure, homology modelling and the amino acid residues that are responsible for proton transfer to the active site are currently unknown making it challenging to provide a detailed rational for these findings. However, these data provide further evidence that this class of underexplored CA deserves more attention.


Assuntos
Aminas/farmacologia , Aminoácidos/farmacologia , Diatomáceas/enzimologia , Aminas/química , Aminoácidos/química , Anidrases Carbônicas/metabolismo , Humanos , Modelos Moleculares
18.
Photosynth Res ; 137(2): 263-280, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29572588

RESUMO

Diatoms are unicellular algae and important primary producers. The process of carbon fixation in diatoms is very efficient even though the availability of dissolved CO2 in sea water is very low. The operation of a carbon concentrating mechanism (CCM) also makes the more abundant bicarbonate accessible for photosynthetic carbon fixation. Diatoms possess carbonic anhydrases as well as metabolic enzymes potentially involved in C4 pathways; however, the question as to whether a C4 pathway plays a general role in diatoms is not yet solved. While genome analyses indicate that the diatom Phaeodactylum tricornutum possesses all the enzymes required to operate a C4 pathway, silencing of the pyruvate orthophosphate dikinase (PPDK) in a genetically transformed cell line does not lead to reduced photosynthetic carbon fixation. In this study, we have determined the intracellular location of all enzymes potentially involved in C4-like carbon fixing pathways in P. tricornutum by expression of the respective proteins fused to green fluorescent protein (GFP), followed by fluorescence microscopy. Furthermore, we compared the results to known pathways and locations of enzymes in higher plants performing C3 or C4 photosynthesis. This approach revealed that the intracellular distribution of the investigated enzymes is quite different from the one observed in higher plants. In particular, the apparent lack of a plastidic decarboxylase in P. tricornutum indicates that this diatom does not perform a C4-like CCM.


Assuntos
Diatomáceas/enzimologia , Diatomáceas/fisiologia , Fotossíntese/fisiologia , Arabidopsis/fisiologia , Ciclo do Carbono , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Mitocôndrias/enzimologia , Fosfoenolpiruvato Carboxilase/classificação , Fosfoenolpiruvato Carboxilase/metabolismo , Piruvato Carboxilase/genética , Piruvato Carboxilase/metabolismo , Zea mays/fisiologia
19.
Arch Biochem Biophys ; 645: 100-106, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29574051

RESUMO

Dimethylsulfoniopropionate (DMSP) is one of the most abundant molecules on earth and plays a pivotal role in the marine sulfur cycle. DMSP is believed to be synthesized from methionine by a four-step reaction pathway in marine algae. The genes responsible for biosynthesis of DMSP remain unidentified. A diatom Thalassiosira pseudonana CCMP1335 is an important component of marine ecosystems and contributes greatly to the world's primary production. In this study, through genome search, in vivo activity and functional studies of cDNA products, a gene encoding Thalassiosira methyltransferase (TpMMT) which catalyzes the key step of DMSP synthesis formation of 4-methylthio-2-hydroxybutyrate (DMSHB) from 4-methylthio-2-oxobutyrate (MTHB), was identified. The amino acid sequence of TpMMT was homologous to the methyltransferase from Phaeodactylum tricornutum CCAP 1055/1, but not the recently identified bacterium gene. High salinity and nitrogen limitation stresses caused the increase of DMSP content and TpMMT protein in Thalassiosira. In addition to TpMMT, the enzyme activities for the first three steps could be detected and enhanced under high salinity, suggesting the importance of four-step DMSP synthetic pathway in Thalassiosira.


Assuntos
Diatomáceas/genética , Diatomáceas/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Compostos de Sulfônio/metabolismo , Sequência de Aminoácidos , Diatomáceas/efeitos dos fármacos , Diatomáceas/enzimologia , Concentração de Íons de Hidrogênio , Metionina/análogos & derivados , Metionina/metabolismo , Metiltransferases/química , Nitrogênio/farmacologia , Salinidade , Estresse Salino/genética , Temperatura Ambiente , Regulação para Cima
20.
J Enzyme Inhib Med Chem ; 33(1): 707-713, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29577755

RESUMO

The inhibition of the δ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii, TweCAδ, was investigated using a panel of 36 mono- and di-thiocarbamates chemotypes that have recently been shown to inhibit mammalian and pathogenic CAs belonging to the α- and ß-classes. TweCAδ was not significantly inhibited by most of such compounds (KI values above 20 µM). However, some aliphatic, heterocyclic, and aromatic mono and di-thiocarbamates inhibited TweCAδ in the low micromolar range. For some compounds incorporating the piperazine ring, TweCAδ was effectively inhibited (KIs from 129 to 791 nM). The most effective inhibitors identified in this study were 3,4-dimethoxyphenyl-ethyl-mono-thiocarbamate (KI of 67.7 nM) and the R-enantiomer of the nipecotic acid di-thiocarbamate (KI of 93.6 nM). Given that the activity and inhibition of this class of enzyme have received limited attention until now, this study provides new molecular probes and information for investigating the role of δ-CAs in the carbon fixation processes in diatoms, which are responsible for significant amounts of CO2 taken from the atmosphere by these marine organisms.


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Diatomáceas/enzimologia , Tiocarbamatos/farmacologia , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Tiocarbamatos/síntese química , Tiocarbamatos/química
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