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1.
Biochem Biophys Res Commun ; 493(1): 58-63, 2017 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-28919416

RESUMO

Aspergillus fumigatus is a human pathogen responsible for deadly infections in immune-compromised patients. A potential strategy for treating A. fumigatus infections is by targeting the biosynthesis of cell wall components, such as galactofuranase, which is absent in humans. Galactofuranose biosynthesis is initiated by the flavoenzyme UDP-galactopyranose mutase (UGM), which converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UGM requires the reduced form of the flavin for activity, which is obtained by reacting with NADPH. We aimed to identify inhibitors of UGM by screening a kinase inhibitor library using ThermoFAD, a flavin fluorescence thermal shift assay. The screening assay identified flavopiridol as a compound that increased the melting temperature of A. fumigatus UGM. Further characterization showed that flavopiridol is a non-competitive inhibitor of UGM and docking studies suggest that it binds in the active site. This compound does not inhibit the prokaryotic UGM from Mycobacteria tuberculosis.


Assuntos
Aspergillus fumigatus/enzimologia , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Flavonoides/química , Transferases Intramoleculares/antagonistas & inibidores , Piperidinas/química , Espectrometria de Fluorescência/métodos , Ativação Enzimática , Flavinas/química , Transferases Intramoleculares/análise , Mapeamento de Interação de Proteínas , Temperatura
2.
J Biol Chem ; 292(11): 4519-4532, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28082677

RESUMO

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mt-tRNAMet, and mt-tRNAPhe, and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in mitochondrial gene expression.


Assuntos
Transferases Intramoleculares/metabolismo , RNA/metabolismo , Linhagem Celular , Humanos , Transferases Intramoleculares/análise , Transferases Intramoleculares/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Interferência de RNA , RNA Mitocondrial , RNA Ribossômico 16S/metabolismo , RNA Interferente Pequeno/genética , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Fenilalanina/metabolismo
3.
Mycorrhiza ; 24(6): 419-30, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24419810

RESUMO

It has been shown in a number of pathosystems that arbuscular mycorrhizal (AM) fungi confer resistance against root pathogens, including in interactions between Medicago truncatula and the root rot-causing oomycete Aphanomyces euteiches. For the current study of these interactions, a split root system was established for plant marker gene analysis in order to study systemic defense responses and to compare them with local interactions in conventional pot cultures. It turned out, however, that split root systems and pot cultures were in different physiological stages. Genes for pathogenesis-related proteins and for enzymes involved in flavonoid biosynthesis were generally more highly expressed in split root systems, accompanied by changes in RNA accumulation for genes encoding enzymes involved in phytohormone biosynthesis. Against expectations, the pathogen showed increased activity in these split root systems when the AM fungus Funneliformis mosseae was present separately in the distal part of the roots. Gene expression analysis revealed that this is associated in the pathogen-infected compartment with a systemic down-regulation of a gene coding for isochorismate synthase (ICS), a key enzyme of salicylic acid biosynthesis. At the same time, transcripts of genes encoding pathogenesis-related proteins and for enzymes involved in the biosynthesis of flavonoids accumulated to lower levels. In conventional pot cultures showing decreased A. euteiches activity in the presence of the AM fungus, the ICS gene was down regulated only if both the AM fungus and the pathogen were present in the root system. Such negative priming of salicylic acid biosynthesis could result in increased activities of jasmonate-regulated defense responses and could explain mycorrhiza-induced resistance. Altogether, this study shows that the split root system does not reflect a systemic interaction between F. mosseae and A. euteiches in M. truncatula and indicates the importance of testing such systems prior to the analysis of mycorrhiza-induced resistance.


Assuntos
Antibiose , Aphanomyces/fisiologia , Glomeromycota/fisiologia , Medicago truncatula/microbiologia , Medicago truncatula/parasitologia , Flavonoides/biossíntese , Perfilação da Expressão Gênica , Transferases Intramoleculares/análise , Transferases Intramoleculares/genética , Redes e Vias Metabólicas/genética , Micorrizas/fisiologia , Raízes de Plantas/microbiologia , Raízes de Plantas/parasitologia , Ácido Salicílico/metabolismo , Fatores de Virulência/análise , Fatores de Virulência/genética
4.
Biosci Biotechnol Biochem ; 71(10): 2543-50, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17928701

RESUMO

Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)-2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)- and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed.


Assuntos
Genes Bacterianos , Transferases Intramoleculares/biossíntese , Methylococcus capsulatus/metabolismo , Células Procarióticas/metabolismo , Esqualeno Mono-Oxigenase/biossíntese , Motivos de Aminoácidos , Sequência de Aminoácidos , Sistema Livre de Células , Cromatografia Gasosa , Clonagem Molecular , Sequência Conservada , Escherichia coli/química , Técnicas In Vitro , Transferases Intramoleculares/análise , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Transferases Intramoleculares/isolamento & purificação , Methylococcus capsulatus/enzimologia , Methylococcus capsulatus/genética , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Plasmídeos , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Esqualeno Mono-Oxigenase/análise , Esqualeno Mono-Oxigenase/química , Esqualeno Mono-Oxigenase/genética , Esqualeno Mono-Oxigenase/isolamento & purificação , Estereoisomerismo
5.
Plant Cell Physiol ; 47(5): 673-7, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16531457

RESUMO

Sterols, essential eukaryotic constituents, are biosynthesized through either cyclic triterpenes, lanosterol (fungi and animals) or cycloartenol (plants). The cDNA for OSC7 of Lotus japonicus was shown to encode lanosterol synthase (LAS) by the complementation of a LAS-deficient mutant yeast and structural identification of the accumulated lanosterol. A double site-directed mutant of OSC7, in which amino acid residues crucial for the reaction specificity were changed to the cycloartenol synthase (CAS) type, produced parkeol and cycloartenol. The multiple amino acid sequence alignment of a conserved region suggests that the LAS of different eukaryotic lineages emerged from the ancestral CAS by convergent evolution.


Assuntos
Células Eucarióticas/metabolismo , Transferases Intramoleculares/fisiologia , Lotus/fisiologia , Fitosteróis/metabolismo , Triterpenos/metabolismo , Sequência de Aminoácidos , Evolução Biológica , DNA de Plantas/análise , DNA de Plantas/genética , Transferases Intramoleculares/análise , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Lanosterol/análogos & derivados , Lanosterol/análise , Lanosterol/química , Lanosterol/metabolismo , Lotus/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fitosteróis/análise , Fitosteróis/química , Saccharomyces cerevisiae/genética
6.
Plant Cell Physiol ; 47(5): 565-71, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16531458

RESUMO

Sterols are important as structural components of plasma membranes and precursors of steroidal hormones in both animals and plants. Plant sterols show a wide structural variety and significant structural differences from those of animals. To elucidate the origin of structural diversity in plant sterols, their biosynthesis has been extensively studied [Benveniste (2004) Annu. Rev. Plant. Biol. 55: 429, Schaller (2004) Plant Physiol. Biochem. 42: 465]. The differences in the biosynthesis of sterols between plants and animals begin at the step of cyclization of 2,3-oxidosqualene, which is cyclized to lanosterol in animals and to cycloartenol in plants. However, here we show that plants also have the ability to synthesize lanosterol directly from 2,3-oxidosqualene, which may lead to a new pathway to plant sterols. The Arabidopsis gene At3g45130, designated LAS1, encodes a functional lanosterol synthase in plants. A phylogenetic tree showed that LAS1 belongs to the previously uncharacterized branch of oxidosqualene cyclases, which differs from the cycloartenol synthase branch. Panax PNZ on the same branch was also shown to be a lanosterol synthase in a yeast heterologous expression system. The higher diversity of plant sterols may require two biosynthetic routes in steroidal backbone formation.


Assuntos
Arabidopsis/fisiologia , Transferases Intramoleculares/fisiologia , Lanosterol/biossíntese , Magnoliopsida/fisiologia , Fitosteróis/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , DNA de Plantas/análise , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas/genética , Transferases Intramoleculares/análise , Transferases Intramoleculares/genética , Magnoliopsida/genética , Dados de Sequência Molecular , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Panax/genética , Panax/fisiologia , Filogenia , Fitosteróis/análise , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Esqualeno/análogos & derivados , Esqualeno/metabolismo
7.
Eukaryot Cell ; 4(6): 1147-54, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15947206

RESUMO

Galactofuranose (Gal(f)) is a novel sugar absent in mammals but present in a variety of pathogenic microbes, often within glycoconjugates that play critical roles in cell surface formation and the infectious cycle. In prokaryotes, Gal(f) is synthesized as the nucleotide sugar UDP-Gal(f) by UDP-galactopyranose mutase (UGM) (gene GLF). Here we used a combinatorial bioinformatics screen to identify a family of candidate eukaryotic GLFs that had previously escaped detection. GLFs from three pathogens, two protozoa (Leishmania major and Trypanosoma cruzi) and one fungus (Cryptococcus neoformans), had UGM activity when expressed in Escherichia coli and assayed in vivo and/or in vitro. Eukaryotic GLFs are closely related to each other but distantly related to prokaryotic GLFs, showing limited conservation of core residues around the substrate-binding site and flavin adenine dinucleotide binding domain. Several eukaryotes not previously investigated for Gal(f) synthesis also showed strong GLF homologs with conservation of key residues. These included other fungi, the alga Chlamydomonas and the algal phleovirus Feldmannia irregularis, parasitic nematodes (Brugia, Onchocerca, and Strongyloides) and Caenorhabditis elegans, and the urochordates Halocynthia and Cionia. The C. elegans open reading frame was shown to encode UGM activity. The GLF phylogenetic distribution suggests that Gal(f) synthesis may occur more broadly in eukaryotes than previously supposed. Overall, GLF/Gal(f) synthesis in eukaryotes appears to occur with a disjunct distribution and often in pathogenic species, similar to what is seen in prokaryotes. Thus, UGM inhibition may provide an attractive drug target in those eukaryotes where Gal(f) plays critical roles in cellular viability and virulence.


Assuntos
Cryptococcus neoformans/genética , Células Eucarióticas/enzimologia , Transferases Intramoleculares/genética , Leishmania major/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Western Blotting , Biologia Computacional , Sequência Conservada , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/patogenicidade , Escherichia coli/genética , Transferases Intramoleculares/análise , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Leishmania major/enzimologia , Leishmania major/patogenicidade , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/patogenicidade
8.
Metab Eng ; 7(3): 165-73, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15885615

RESUMO

Previous studies revealed that two genes-sanU and sanV were associated with nikkomycin biosynthesis in Streptomyces ansochromogenes. A plasmid used to increase an extra copy of sanU and sanV was constructed and introduced into wild-type strain. HPLC results showed that nikkomycin production of recombinant strain was about 1.8 fold than that of wild-type strain. RT-PCR analysis indicated that the transcriptional level of sanU and sanV in this recombinant strain was about two folds than that of wild-type strain. The sanU and sanV were expressed in E. coli BL21 (DE3). SanU and SanV were purified individually. SanU and SanV assembled with coenzyme B12 to form a complete enzyme in vitro, which showed glutamate mutase activity. The glutamate mutase converted L-glutamate toL-threo-beta-Methylaspartic acid, and then l-threo-beta-Methylaspartic acid was probably deaminated to form 2-oxo-3-methylsuccinic acid to join biosynthetic pathway of the peptidyl moiety HPHT in S. ansochromogenes. SanU is the coenzyme B12-binding component and more than two folds of SanU are required for maximal enzyme activity. The optimal pH and temperature for the formed enzyme are 7.5-8.5 and 35-42 degrees C, respectively. Sulfhydryl compounds are important for activity of the reassembled enzyme.


Assuntos
Aminoglicosídeos/biossíntese , Melhoramento Genético/métodos , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Engenharia de Proteínas/métodos , Streptomyces/genética , Streptomyces/metabolismo , Aminoglicosídeos/genética , Ativação Enzimática , Estabilidade Enzimática , Dosagem de Genes , Transferases Intramoleculares/análise , Transferases Intramoleculares/genética , Especificidade da Espécie , Streptomyces/classificação
9.
Chem Biol ; 11(1): 121-6, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15113001

RESUMO

The membrane protein squalene-hopene cyclase was cocrystallized with 2-azasqualene and analyzed by X-ray diffraction to 2.13 A resolution. The conformation of this close analog was clearly established, and it agreed with the common textbook presentation. The bound squalene undergoes only small conformational changes during the formation of rings A through D, thus requiring no intermediate. However, ring E formation is hindered by an entropic barrier, which may explain its absence in the steroids. The structure analysis revealed a mobile region between the active center cavity and the membrane, which may melt, opening a passage for squalene and hopene.


Assuntos
Transferases Intramoleculares/química , Proteínas de Membrana/química , Esqualeno/química , Triterpenos/síntese química , Bacillaceae/enzimologia , Sítios de Ligação , Catálise , Membrana Celular/metabolismo , Cristalografia por Raios X , Ciclização , Transferases Intramoleculares/análise , Transferases Intramoleculares/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Modelos Moleculares , Conformação Proteica , Esqualeno/análogos & derivados
10.
Org Lett ; 6(5): 803-6, 2004 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-14986979

RESUMO

1-methylidenesqualene and 25-methylidenesqualene were converted to 30-methylidenehop-22(29)-ene by squalene:hopene cyclase from Alicyclobacillus acidocaldarius. It was remarkable that both analogues generated the same product. The hopanyl intermediate cation, stabilized by the methylidene residue, enabled a rotation of the isobutenyl group at C-21 prior to the final proton elimination. In contrast, in the formation of hop-22(29)-ene, the final proton abstraction takes place regiospecifically from the Z-methyl group, which was verified by cyclization of (1,1,1,24,24,24-(2)H(6))squalene into (23,23,23,30,30,30-(2)H(6))hop-22(29)-ene. [reaction: see text]


Assuntos
Bacillus/enzimologia , Bacilos Gram-Positivos Formadores de Endosporo/enzimologia , Transferases Intramoleculares/análise , Sondas Moleculares , Esqualeno/síntese química , Sítios de Ligação , Estrutura Molecular , Esqualeno/análogos & derivados , Esqualeno/metabolismo
11.
J Biol Chem ; 276(37): 34934-40, 2001 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-11406626

RESUMO

To characterize the substrate specificity of the putative RNA:pseudouridine (Psi)-synthase encoded by the Saccharomyces cerevisiae open reading frame (ORF) YGR169c, the corresponding gene was deleted in yeast, and the consequences of the deletion on tRNA and small nuclear RNA modification were tested. The resulting DeltaYGR169c strain showed no detectable growth phenotype, and the only difference in Psi formation in stable cellular RNAs was the absence of Psi at position 31 in cytoplasmic and mitochondrial tRNAs. Complementation of the DeltaYGR169c strain by a plasmid bearing the wild-type YGR169c ORF restored Psi(31) formation in tRNA, whereas a point mutation of the enzyme active site (Asp(168)-->Ala) abolished tRNA:Psi(31)-synthase activity. Moreover, recombinant His(6)-tagged Ygr169 protein produced in Escherichia coli was capable of forming Psi(31) in vitro using tRNAs extracted from the DeltaYGR169c yeast cells as substrates. These results demonstrate that the protein encoded by the S. cerevisiae ORF YGR169c is the Psi-synthase responsible for modification of cytoplasmic and mitochondrial tRNAs at position 31. Because this is the sixth RNA:Psi-synthase characterized thus far in yeast, we propose to rename the corresponding gene PUS6 and the expressed protein Pus6p. Finally, the cellular localization of the green fluorescent protein-tagged Pus6p was studied by functional tests and direct fluorescence microscopy.


Assuntos
Transferases Intramoleculares/análise , Saccharomyces cerevisiae/enzimologia , Citoplasma/metabolismo , Hidroliases , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Mitocôndrias/metabolismo , Fases de Leitura Aberta , Pseudouridina/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Especificidade por Substrato
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