Isoleucyl-tRNA synthetase genes from the yeast Saccharomyces cerevisiae and the ciliated protozoan Tetrahymena thermophila were sequenced. The intronless S. cerevisiae gene (ILS1) encodes a putative polypeptide of 1072 amino acids. Two putative promoter elements were identified, one for general amino acid control and one for constitutive transcription. A heat shock protein gene lies upstream of ILS1. The T. thermophila isoleucyl-tRNA synthetase gene (ilsA: formerly cupC) has eight introns, four transcription start sites, and codes for a putative polypeptide of 1081 amino acids with two leucine-zippers. These eukaryotic isoleucyl-tRNA synthetases are 47% identical. They are compared to homologous enzymes from Escherichia coli and an archaebacterium, and to other aminoacyl-tRNA synthetases. / Intron sequences and junctions from T. thermophila and other eukaryotes were analyzed and all but yeast and mammalian introns were found to be A + T enriched. T. thermophila transcription start sites were analyzed and occur at a T or an A within the consensus sequence (A/T)$ sb{ rm n}$ T A A (A)$ sb{ rm n}.$

No description available.

A biomassa vegetal é a maior fonte de carbono renovável disponível, e é composta de celulose, hemicelulose e lignina. A lignina, um heteropolímero polifenólico complexo, é uma barreira a utilização mais eficiente da biomassa. Fungos desenvolveram formas de superar o obstáculo imposto pela lignina, através da atividade de enzimas como peroxidases, e da ação de pequenas moléculas reativas. As duas formas dependem da presença de peróxido de hidrogênio, que é produzido pela atividade de enzimas como aril-álcool oxidases (AAOs). AAOs são enzimas pertencentes a superfamília GMC de oxidoredutases e, portanto, possuem FAD como grupo prostético. Realizam a conversão de álcoois aromáticos à aldeídos, com concomitante produção de peróxido de hidrogênio. Devido à capacidade de oxidação de substratos variados, e dependente somente de oxigênio molecular, AAOs têm potencial aplicação na indústria biotecnológica, tanto como produtoras de peróxido de hidrogênio quanto na produção de químicos-plataforma a partir da biomassa. No entanto, AAOs ainda têm poucos exemplares caracterizados, e a maior parte dos estudos publicados é referente a enzimas do gênero de fungos basidiomicetos Pleurotus sp.. A enzima MtAAOx do fungo ascomiceto termofílico M. thermophila foi produzida de forma heteróloga em A. nidulans, e algumas de suas propriedades bioquímicas e estruturais foram determinadas. MtAAOx é uma enzima extracelular monomérica glicosilada, com grupo FAD dissociável, e capaz de oxidar álcoois aromáticos fenólicos e não fenólicos, incluindo um monômero da lignina, coniferil álcool, substrato para o qual a enzima teve mais afinidade. MtAAOx, além de oxidar álcoois aromáticos, também foi capaz de oxidar um substrato heterocíclico, 5-HMF, e de utilizar outro aceptor de elétrons além do oxigênio. Esses resultados, aliados a baixa similaridade entre sequencias de aminoácidos indicam que MtAAOx tem sítio catalítico e canal de acesso do substrato distintos das AAOs até o momento caracterizadas. A enzima MtAAOx deglicosilada teve estabilidade térmica reduzida em comparação com a enzima nativa. A atividade enzimática foi afetada positivamente com a presença de alguns cátions, entre eles Ca2+. Além de afetar a atividade, Ca2+proporcionou ganho de estabilidade térmica, com aumento da temperatura de melting em 5 ºC. Esta enzima é uma nova aril-álcool oxidase caracterizada, a primeira de um fungo ascomiceto, e acredita-se que tenha papel na degradação da lignina da biomassa vegetal. / Plant biomass is the largest source of available renewable carbon, and is composed of cellulose, hemicellulose and lignin. Lignin, a complex polyphenolic heteropolymer, is a barrier to a more efficient use of biomass. Fungi have developed ways to overcome the obstacle imposed by lignin through the activity of enzymes such as peroxidases and the action of small reactive molecules. The two forms depend on the presence of hydrogen peroxide, which is produced by the activity of enzymes such as aryl-alcohol oxidases (AAOs). AAOs are enzymes belonging to the GMC superfamily of oxidoreductases and therefore have FAD as the prosthetic group. They perform the conversion of aromatic alcohols to aldehydes, with simultaneous production of hydrogen peroxide. Because of their capacity of oxidizing various substrates, and dependence only on molecular oxygen, AAOs have potential applications in the biotech industry, both as hydrogen peroxide producers and in the production of platform chemicals derived from biomass. However, characterized AAOs are few, most of the published studies being on enzymes of basidiomycete fungi from genus Pleurotus sp.. The MtAAOx enzyme of the thermophilic ascomycete M. thermophila was produced heterologously in A. nidulans, and some of its biochemical and structural properties were determined. MtAAOx is a glycosylated, monomeric, extracellular enzyme with a dissociable FAD group, capable of oxidizing phenolic and non-phenolic aromatic alcohols, including a lignin monomer, coniferyl alcohol, the substrate for which the enzyme has the highest affinity. MtAAOx, besides oxidizing aromatic alcohols, was also able to oxidize a heterocyclic substrate, 5-HMF, and to use another electron acceptor in addition to oxygen. These results, combined with the low similarity between amino acid sequences, indicate that MtAAOx has a catalytic site and substrate access channel distinct from characterized AAOs\'. The deglycosylated MtAAOx enzyme had reduced thermal stability compared to the native enzyme. The enzymatic activity was positively affected by the presence of some cations, including Ca2+. In addition to affecting the activity, Ca2+ improved thermal stability, with 5 °C increase of the melting temperature. This enzyme is a novel aryl-alcohol oxidase characterized, the first of an ascomycete fungus, and is believed to play a role in the lignin degradation of plant biomass.

A biomassa vegetal é a maior fonte de carbono renovável disponível, e é composta de celulose, hemicelulose e lignina. A lignina, um heteropolímero polifenólico complexo, é uma barreira a utilização mais eficiente da biomassa. Fungos desenvolveram formas de superar o obstáculo imposto pela lignina, através da atividade de enzimas como peroxidases, e da ação de pequenas moléculas reativas. As duas formas dependem da presença de peróxido de hidrogênio, que é produzido pela atividade de enzimas como aril-álcool oxidases (AAOs). AAOs são enzimas pertencentes a superfamília GMC de oxidoredutases e, portanto, possuem FAD como grupo prostético. Realizam a conversão de álcoois aromáticos à aldeídos, com concomitante produção de peróxido de hidrogênio. Devido à capacidade de oxidação de substratos variados, e dependente somente de oxigênio molecular, AAOs têm potencial aplicação na indústria biotecnológica, tanto como produtoras de peróxido de hidrogênio quanto na produção de químicos-plataforma a partir da biomassa. No entanto, AAOs ainda têm poucos exemplares caracterizados, e a maior parte dos estudos publicados é referente a enzimas do gênero de fungos basidiomicetos Pleurotus sp.. A enzima MtAAOx do fungo ascomiceto termofílico M. thermophila foi produzida de forma heteróloga em A. nidulans, e algumas de suas propriedades bioquímicas e estruturais foram determinadas. MtAAOx é uma enzima extracelular monomérica glicosilada, com grupo FAD dissociável, e capaz de oxidar álcoois aromáticos fenólicos e não fenólicos, incluindo um monômero da lignina, coniferil álcool, substrato para o qual a enzima teve mais afinidade. MtAAOx, além de oxidar álcoois aromáticos, também foi capaz de oxidar um substrato heterocíclico, 5-HMF, e de utilizar outro aceptor de elétrons além do oxigênio. Esses resultados, aliados a baixa similaridade entre sequencias de aminoácidos indicam que MtAAOx tem sítio catalítico e canal de acesso do substrato distintos das AAOs até o momento caracterizadas. A enzima MtAAOx deglicosilada teve estabilidade térmica reduzida em comparação com a enzima nativa. A atividade enzimática foi afetada positivamente com a presença de alguns cátions, entre eles Ca2+. Além de afetar a atividade, Ca2+proporcionou ganho de estabilidade térmica, com aumento da temperatura de melting em 5 ºC. Esta enzima é uma nova aril-álcool oxidase caracterizada, a primeira de um fungo ascomiceto, e acredita-se que tenha papel na degradação da lignina da biomassa vegetal. / Plant biomass is the largest source of available renewable carbon, and is composed of cellulose, hemicellulose and lignin. Lignin, a complex polyphenolic heteropolymer, is a barrier to a more efficient use of biomass. Fungi have developed ways to overcome the obstacle imposed by lignin through the activity of enzymes such as peroxidases and the action of small reactive molecules. The two forms depend on the presence of hydrogen peroxide, which is produced by the activity of enzymes such as aryl-alcohol oxidases (AAOs). AAOs are enzymes belonging to the GMC superfamily of oxidoreductases and therefore have FAD as the prosthetic group. They perform the conversion of aromatic alcohols to aldehydes, with simultaneous production of hydrogen peroxide. Because of their capacity of oxidizing various substrates, and dependence only on molecular oxygen, AAOs have potential applications in the biotech industry, both as hydrogen peroxide producers and in the production of platform chemicals derived from biomass. However, characterized AAOs are few, most of the published studies being on enzymes of basidiomycete fungi from genus Pleurotus sp.. The MtAAOx enzyme of the thermophilic ascomycete M. thermophila was produced heterologously in A. nidulans, and some of its biochemical and structural properties were determined. MtAAOx is a glycosylated, monomeric, extracellular enzyme with a dissociable FAD group, capable of oxidizing phenolic and non-phenolic aromatic alcohols, including a lignin monomer, coniferyl alcohol, the substrate for which the enzyme has the highest affinity. MtAAOx, besides oxidizing aromatic alcohols, was also able to oxidize a heterocyclic substrate, 5-HMF, and to use another electron acceptor in addition to oxygen. These results, combined with the low similarity between amino acid sequences, indicate that MtAAOx has a catalytic site and substrate access channel distinct from characterized AAOs\'. The deglycosylated MtAAOx enzyme had reduced thermal stability compared to the native enzyme. The enzymatic activity was positively affected by the presence of some cations, including Ca2+. In addition to affecting the activity, Ca2+ improved thermal stability, with 5 °C increase of the melting temperature. This enzyme is a novel aryl-alcohol oxidase characterized, the first of an ascomycete fungus, and is believed to play a role in the lignin degradation of plant biomass.

Charles University in Prague Faculty of Pharmacy in Hradec králové Department of Pharmaceutical Botany and Ecology Author: Lenka Nechanická Supervisor: RNDr. Jitka Vytlačilová, Ph.D. Title of diploma thesis: Antiprotozoal activity of natural substances The discovery of new active substances and plants with a potential antiprotozoal effect nowadays is the aim of many studies and is required for obtaining more active drugs in a number of protozoal disease. In this study was investigated antiprotozoal activity of extracts obtained from various plant parts Salvia officinalis, Apium graveolens L. var. rapaceum, Evodia rutaecarpa, Coptis chinensis, Zanthoxylum nitidum and Ziziphus jujuba. Effect of the tested extracts was evaluated in a typical unicellular organism Tetrahymena thermophila using MTT method. From the values obtained the percent inhibition was detected Tetrahymena thermophila and for each extract value calculated median inhibitory concentrations IC50. Of the extracts tested had the greatest antiprotozoal activity of the extract of C. chinensis, further extracts activity decreases in the order C. chinensis > Z. nitidum > Z. jujuba > S. officinalis > E. rutaecarpa > A. graveolens L. var. rapaceum Key words: antiprotozoal activity, Tetrahymena thermophila, cytotoxicity, natural substances

No description available.

The CO dehydrogenase~linked ferredoxin from acetate-grown <i>Methanosarcina thermophiIa</i> was characterized to determine the structure and biochemical properties of the iron-sulfur clusters. Chemical and spectroscopic analyses indicated that the ferredoxin contained two [4Fe-4S] clusters per monomer of 6,790 Da, although a [3Fe-4S] species was also detected in the oxidized protein. The midpoint potentials of the [4Fe-4S] and [3Fe~4S] clusters at pH 7 were -407 m V and + 103 m V, respectively. Evidence from biochemical and spectroscopic studies indicated that the [3Fe-4S] species may have been formed from [4Fe-4S] clusters when ferredoxin was oxidized. The gene encoding the CO dehydrogenase-linked ferredoxin (<i>fdxA</i>) in <i>Ms. thermophila</i> had the coding capacity for a 6,230-Da protein which contained eight cysteines with spacings typical of 2[4Fe-4S] ferredoxins. A second open reading frame (ORF1) was also identified which had the potential to encode a 2[4Fe-4S] bacterial-like ferredoxin (5,850 Da). The deduced proteins from <i>fdxA</i> and ORF1 were 62% identical. <i>fdxA</i> and ORFI were present as single copies in the genome and each was transcribed on a monocistronic mRNA. Both <i>fdxA</i> and ORF1 were transcribed in cells grown on methanol and trimethylamine, but only the <i>fdxA</i> -specific transcript was detected in acetate-grown cells. The apparent transcriptional start sites of <i>fdxA</i> and ORFI were downstream of sequences which had high identity with the consensus methanogen promoter. The heterodisulfide of two cofactors unique to the methanogenic microorganisms, HS-HTP and HS-CoM, was enzymatically reduced in cell extracts of <i>Ms. thermophila</i> using electrons from the oxidation of either H₂ or CO. The homodisulfides of either HS-HTP or HS-CoM were not reduced under the same conditions. The results indicated that methane is formed by reductive demethylation of CH₃-S-CoM using HS-HTP as a reductant in <i>Ms. thermophila</i>. Coupling of CO oxidation with reduction of the heterodisulfide suggested that the CO dehydrogenase-linked ferredoxin may be involved, although the details of electron flow are not known. / Ph. D.

The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila was examined by two-dimensional polyacrylamide gel electrophoresis to determine the extent of regulation by the growth substrate. More than 100 mutually-exclusive spots were present in acetate- and methanol-grown cells suggesting a high degree of regulation. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the nickel-containing carbon monoxide dehydrogenase (CODH) complex were identified in acetate-grown cells. The nickel-containing methyl coenzyme M methylreductase from acetate-grown M. thermophila was purified 16-fold from a cell extract to apparent homogeneity. The enzyme had a native molecular weight of between 132,000 and 141,000 and contained three subunits with a configuration of a1B1y1-. The as-isolated enzyme was inactive, but could be reductively reactivated by either titanium (III) citrate or reduced ferredoxin. Reactivation with ferredoxin was a simplification over previously reported reactivation systems. ATP stimulated, but was not required for reactivation. The CO dehydrogenase enzyme complex from M. thermophila was purified and separated into its respective components: the CO-oxidizing nickel/iron-sulfur (Ni/Fe-S) component and the cobalt-containing corrinoid/iron sulfur (Co/Fe-S) component. EPR spectroscopy and spectroelectrochemical titration of the Fe-S centers of the Ni/Fe-S component indicated the presence of two low-potential [4Fe-4S]2+/1+ centers and third high-potential center whose Fe-S configuration is unknown. When reduced with CO, the NilFe-S component exhibited a previously unobserved Ni-Fe-C EPR signal. The Co/Fe-S component contained one [4Fe-4S]2+/1+ cluster, and the as-isolated corrinoid in the component was in the base-off conformation suggesting that modulation of the electron density of the cobalt ion may result in a modified reactivity of the active site of the corrin. The CODH enzyme complex and isolated Co/Fe-S component reductively dechlorinated trichloroethylene to cis-dichloroethylene, trans-dichloroethylene, 1,1-dichloroethylene, vinyl chloride, and ethylene. Factor III also catalyzed the dechlorination of trichloroethylene when in the presence of titanium (III) citrate. Reconstitution of the Co/Fe-S component with the CO-reduced NilFe-S component also allowed dechlorination demonstrating an electron transfer from the reduced Ni/Fe-S component to the Co/Fe-S component. / Ph. D.

Methanosarcina are methanogens capable of growth and methanogenesis from H₂/CO₂, formate, methanol, methylamines, and acetate. Methanosarcina conserve energy by coupling electron transport and methyl transfer to the generation of ion gradients during acetoclastic growth. This work focuses on cytochrome b and heterodisulfide reductase, two proteins involved in energy conservation by electron transport. A procedure was developed for mass cultivation of Methanosarcina thermophila strain TM-1 in 12-liter fermentations which produced up to 10 grams wet weight/liter, in order to facilitate biochemical studies. Cytochromes occurring in Methanosarcina thermophila were characterized spectrophotometrically using chemical and physiological reactants. This analysis revealed two heme centers, one of which was only reduced by Na₂S₂O₄ or carbon monoxide. Partially purified cytochromes were found to be present in a complex and were characterized by electrophoretic and spectrophotometric analysis. The cytochrome-containing protein was found to contain two hemes and had an M_r of 28,000 Da. Heterodisulfide reductase was isolated from the soluble fraction by anion exchange chromatography and assayed using methyl viologen as an artificial electron donor. Electron transport from CO to the heterodisulfide of 2-mercaptoethanesulfonic acid (HS-CoM) and 7- mercaptoheptanoylthreonine phosphate (HS-HTP) was reconstituted using carbon monoxide dehydrogenase, ferredoxin, membranes, and heterodisulfide reductase. Both membranes and ferredoxin were required for reduction of the heterodisulfide. / Master of Science

The genes encoding the acetate kinase and phosphotransacetylase enzymes from <i>Methanosarcina thermophila</i> were isolated from a genomic library on a fifteen kilobase fragment The genes are located adjacent to one another, with the phosphotransacetylase gene (<i>pta</i>) directly upstream of the acetate kinase gene (<i>ack</i>). The two genes were sequenced, along with a third Open Reading Frame (designated <i>orfY</i>). The <i>orfY</i> gene appears to encode a novel protein whose physiological function has yet to be determined. / Ph. D.