Universiẗat, Diss., 2004--Frankfurt (Main).

Thermus bacteria are of special interest because of their ability to live in high temperature environments. Their enzymes exhibit higher and stable activity in industry as compared to mesophilic or synthetic counterparts. Thermus bacteria are capable of reducing heavy metals which is essential in eradication of heavy metal pollution and controlling global warming. Genome rearrangements were investigated in Thermus species and the extent to which they affected the distribution of functionally related genes on the chromosome and its implication on the coherence of the metabolic network. The contribution of horizontal gene transfer to genome rearrangements and the shuffling of genes on the chromosome were analysed. Horizontally transferred genes were identified alongside their donors and recipients, their age and relative time of insertion. Metabolic networks were clustered and compared to determine the extent to which they were affected by rearrangements as a measure of evolutionary pressures experienced by organisms. Factors that enhance protein thermostability were analysed by determining dominant substitutions for amino acid residues and their properties. Protein thermostability was measured using the UNAFold algorithm. Amino acid substitutions were compared between less and highly thermophilic orthologous sequences in T. scotoductus SA-01 and T. thermophilus HB27 respectively. Protein structures were modelled for orthologs that met a defined selection criterion. Dominant amino acid substitutions were analysed in the structures to determine their locations. The contribution of dominant substitutions to energy changes in structures was analysed using FoldX program. Results revealed a uniform distribution of functionally related genes among thermophilic and mesophilic organisms. The contribution of horizontal gene transfer to genome rearrangements was found to be insignificant. Metabolic networks for Thermus species were poorly clustered in correlation with their optimum environmental growth temperatures. Non-polar, small and charged amino acids were found to significantly enhance thermostability. Higher occurrence of alanine substituted by serine and threonine; and arginine substituted by glutamine and lysine were observed to influence thermostability. Structural comparison showed that mutations were mostly located on the surfaces and helices of structures. The positions of mutations appeared to influence their energy contribution to the overall structure as measured by FoldX algorithm. / Thesis (PhD)--University of Pretoria, 2013. / gm2014 / Biochemistry / unrestricted

The aim of this work was focused on study of influence of cultivation conditions on growth of thermophilic bacteria Thermus aquaticus, Thermus scotoductus, Geobacillus thermodenitrificans and Geobacillus thermocatenulatus in cultivation medium recommended by Czech Collection of Microorganisms (CCM). The change of concentration of biomass during cultivation with various pH of media, cultivation temperature, agitation rate and with addition of glucose to medium during cultivation in Erlenmayer flasks was observed. Results served for determination of optimal growth conditions for each microorganism. After optimal growth conditions were obtained, cultivations in fermenter were performed. Growth curves were made from values of absorbance and dry weight of samples.

Universiẗat, Diss., 2007--Frankfurt (Main). / Zsfassung in dt. und engl. Sprache.

Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular. / La 4-amino-5-hidroximetil-2-metilpirimidina quinasa (HMPK, EC 2.7.1.49) es una enzima perteneciente a la superfamilia riboquinasa y participa en la biosíntesis de tiamina (vitamina B1) en bacterias. Se ha descrito que esta enzima es capaz de catalizar dos fosforilaciones consecutivas dependientes de ATP altamente específicas sobre el sustrato hidroximetil pirimidina (HMP), generando como producto hidroximetil pirimidina pirofosfato. Esto contrasta notablemente con lo que se ha observado en las piridoxal quinasas de bacterias Gram positivas (HMPK/PLK, EC 2.7.1.35), un grupo de enzimas homólogas cercanas capaces de fosforilar hidroximetil pirimidina, piridoxal, piridoxina y piridoxamina, pero incapaces de catalizar dos fosforilaciones consecutivas, por lo que sólo producen hidroximetil pirimidina fosfato. Las HMPKs no han sido estudiadas tan exhaustivamente como las HMPK/PLKs y sólo hay dos caracterizaciones breves disponibles en la literatura; la de la HMPK de Escherichia coli y la de Bacillus subtilis. Por lo tanto, aún no se conoce si las propiedades observadas en las enzimas descritas son ubicuas para linajes bacterianos distintos, especialmente aquellos filogenéticamente distantes y que han sido sometido a presiones selectivas fuertes, como los extremófilos. Por esta razón, en este trabajo se realizó la caracterización bioquímica de la HMPK de la enterobacteria Salmonella typhimurium (StHMPK) y de la bacteria termófila Thermus thermophilus (TtHMPK). A través de experimentos de estequiometría de reacción y análisis de generación de productos, se demostró que ambas enzimas son capaces de catalizar dos fosforilaciones consecutivas. Experimentos de especificidad de sustrato revelaron que ambas enzimas son altamente específicas por hidroximetil pirimidina. Análisis filogenéticos mostraron que estas enzimas están estrechamente relacionadas con las HMPKs/PLK de organismos gram positivos, y estas últimas parecen ser descendientes directos de las HMPKs. Por lo tanto, para estudiar cómo estos grupos de enzimas han divergido en términos de sus actividades catalíticas, se realizaron simulaciones de dinámica molecular del complejo ternario (Mg·ATP - HMP) de StHMPK, para analizar el sitio de unión a sustrato y compararlo con el de la HMPK/PLK de Staphylococcus aureus (SaPLK). Los resultados mostraron que existe un alto grado de conservación entre ambos sitios, existiendo sólo unas pocas diferencias que podrían explicar la divergencia funcional observada, principalmente la presencia de una treonina adyacente a la base catalítica en StHMPK, que es reemplazada por una alanina en SaPLK, y la presencia de una glutamina en StHMPK que forma puentes de hidrógeno con el HMP. La caracterización cinética de StHMPK y TtHMPK mostró que ambas enzimas poseen una KM similar para HMP (cercana a 30μM) y que la Vmax para TtHMPK es un orden de magnitud menor que para StHMPK a 37 °C. Sin embargo, estos parámetros fueron obtenidos para las curvas de saturación de HMP, las cuales mostraban un comportamiento del tipo Michaelis-Menten, mientras que las curvas de saturación para ATP mostraron una clara desviación de este modelo y por lo tanto, no se pudieron determinar parámetros cinéticos. Finalmente, se realizó una caracterización estructural y biofísica para evaluar diferencias de estabilidad. Ambas enzimas parecen ser monómeros en las condiciones estudiadas, a diferencia de lo reportado para la enzima de E. coli que forma un tetrámero. Experimentos de desplegamiento por temperatura y agentes químicos mostraron que TtHMPK es significativamente más estable que StHMPK. Las bases estructurales de estas diferencias fueron analizadas mediante simulaciones de dinámica molecular, las que revelaron que la proteína termoestable es más rígida, tiene un menor contenido de residuos polares en el núcleo y tiene mayor cantidad de interacciones electrostáticas que su homólogo mesoestable. / 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase (HMPK, EC 2.7.1.49) is a bacterial enzyme that belongs to the ribokinase superfamily and participates in the thiamine (vitamine B1) biosynthetic pathway. It has been described that this enzyme is capable to catalyze two consecutive highly specific ATP dependent phosphorylations on the substrate hydroxymethyl pyrimidine, yielding hydroxymethyl pyrimidine pyrophosphate. This contrast notoriously with what has been observed for the closely related homologous enzymes pyridoxal kinases from Gram positive bacteria (HMPK/PLK, EC 2.7.1.35), which can phosphorylate hydroxymethyl pyrimidine, pyridoxal, pyridoxine and pyridoxamine, but are unable to catalyze two consecutive phosphorylations, thus only produce hydroxymethyl pyrimidine phosphate. HMPKs have not been as extensively studied as HMPKs/PLK, and only two brief biochemical characterizations are available on the literature; the characterization of the HMPK from Escherichia coli and from Bacillus subtilis. Therefore, it is still unknown whether the properties observed in the described enzymes are ubiquitous among different bacterial lineages, especially those that come from a very distinct phylogenetic background and have been subject to strong selective pressures, as the enzymes from extremophilic organisms. For this reason, in this work we address the biochemical characterization of the HMPK from the enterobacteria Salmonella typhimurium (StHMPK) and the thermophilic bacteria Thermus thermophilus (TtHMPK). Through stoichiometric experiments and product generation analysis, it was established that both enzymes are able to perform two consecutive phosphorylations. Substrate specificity experiments revealed that both enzymes are highly specific for hydroxymethyl pyrimidine. Phylogenetic analysis of these enzymes showed that are closely related to HMPKs/PLK from Gram positive organisms, being the later a direct descendant from HMPKs. Therefore, to study how these two groups of enzymes have diverged so much in terms of their catalytic activities, we analysed the substrate binding site of StHMPK by molecular dynamics simulations of the ternary complex (Mg·ATP - HMP) and compared it to the binding site of the PLK from Staphylococcus aureus (SaPLK). The results showed that there is an overall great conservation among the active sites, with just a few differences that could be responsible for the functional divergences observed, mainly the presence of a threonine residue adjacent to the catalytic base in StHMPK which is replaced by an alanine in SaPLK, and the presence of a glutamine that forms hydrogen bonds with the HMP in StHMPK. Kinetic characterization of StHMPK and TtHMPK showed that both enzymes have a similar KM for HMP (around 30 μM) while the Vmax for TtHMPK is one order of magnitude lower than the Vmax for StHMPK. However, these parameters were obtained only for HMP saturation curves, which showed a Michaelis-Menten behaviour, whereas ATP saturation curves displayed a clear deviation from a Michaelis-Menten model and therefore, no kinetic parameters could be deduced from these experiments. Finally, a biophysical and structural characterization to assess stability differences was performed. Both enzymes seem to be in monomeric state under the conditions assayed, in contrast with what was reported for the enzyme from E. coli, which forms a tetramer. Thermal and chemical unfolding experiments showed that TtHMPK is significantly more stable than StHMPK. The structural basis for these differences were investigated through molecular dynamics simulations, which revealed that the thermostable protein is more rigid, has a reduced content of polar amino acids in its core, and has more electrostatic interactions than its mesostable homologous. / Julio del 2019

No description available.

No description available.

La traduction implique l’utilisation d’aminoacyl-ARNt produits par les aminoacyl-ARNt synthétases (aaRS). Il devrait exister 20 aaRS, une spécifique de chaque acide aminé. Or, les données actuelles montrent qu’une grande majorité des organismes ne possèdent pas l’asparaginyl- (AsnRS) et/ou la glutaminyl-ARNt synthétase (GlnRS). Ils ne peuvent synthétiser l’Asn-ARNtAsn et le Gln-ARNtGln que par l’utilisation de voies impliquant la formation préalable d’aspartyl-ARNtAsn et/ou de glutamyl-ARNtGln. Ces précurseurs « mésacylés » sont synthétisés par une aspartyl-ARNt synthétase et/ou une glutamyl-ARNt synthétase non-discriminantes (AspRS-ND ou GluRS-ND). Ils sont ensuite amidés par une amidotransférase (AdT), pour fournir à la cellule l’Asn-ARNtAsn et/ou le Gln-ARNtGln nécessaires à la traduction des codons Asn et Gln.Ce travail de thèse, effectué dans le contexte biologique de deux organismes différents, Thermus thermophilus et Helicobacter pylori, a permis de montrer que les étapes enzymatiques – formation du précurseur, et amidation par l’AdT – sont réalisées au sein de complexes ribonucléoprotéiques, réunissant l’aaRS-ND, l’ARNtAsn ou l’ARNtGln, et l’AdT : l’Asn-transamidosome ou le Gln-transamidosome. Selon leur origine ou la voie à laquelle ils appartiennent (asparaginylation ou glutaminylation), ces complexes possèdent des particularités mécanistiques et structurales très différentes, mais sont tous adaptés pour éviter la libération des intermédiaires mésacylés toxiques par des stratégies spécifiques. Ce travail permet de mieux comprendre les mécanismes évolutifs qui ont conduit à l’incorporation de l’Asn et de la Gln dans le code génétique. / Protein synthesis requires the biosynthesis of aminoacyl-tRNAs by aminoacyl-tRNA synthétases (aaRS). Since 20 amino acids are présent within the genetic code, 20 aaRS should be used by a single organism. However, the vast majority of organisms found today are deprived of asparaginyl- and/or glutaminyl-tRNA synthetases (Asn- or GlnRS). They can only synthesize Asn-tRNAAsn and/or Gln-tRNAGln through biosynthesis pathways involving the preliminary formation of aspartyl-tRNAAsn and /or glutamyl-tRNAGln. Those « misacylated » precursors are synthesized by so called non-discriminating aspartyl- or glutamyl-tRNA synthetases (ND-AspRS or –GluRS). Then, they are transferred to an amidotransferase (AdT) to provide the Asn-tRNAAsn and/or Gln-tRNAGln species (necessary to fuel protein synthesis) through amidation.This work was performed in the context of two organisms – Thermus thermophilus and Helicobacter pylori. It showed that the two enzymatic steps of asparaginylation and glutaminylation – biosynthesis of the misacylated precursor and amidation by AdT – are carried out within a single ribonucleoprotein complex, namely the (Asn- or Gln-) transamidosome, gathering the ND-aaRS necessary for the misacylation, the tRNA substrate (Asn or Gln) and the AdT. According to their origin or the pathway they originate from (asparaginylation or glutaminylation), those complexes display significant mechanistical and structural peculiarities, but they are all adapted to prevent libération of the toxic misacylated species through specific strategies. This work shed new light on the évolutive mechanisms that led to the incorporation of Asn or Gln into the genetic code.

Diss. med. dent. Zürich. / Literaturverz.

Universiẗat, Diss., 2006--Frankfurt (Main).