Expanded Monomeric Intermediate upon Cold and Heat Unfolding of Phosphofructokinase-2 from Escherichia coli

AbstractFolding studies have been focused mainly on small, single-domain proteins or isolated single domains of larger proteins. However, most of the proteins present in biological systems are composed of multiple domains, and to date, the principles that underlie its folding remain elusive. The unfolding of Pfk-2 induced by GdnHCl has been described by highly cooperative three-state equilibrium (N2↔2I↔2U). This is characterized by a strong coupling between the subunits’ tertiary structure and the integrity of the dimer interface because “I” represents an unstructured and expanded monomeric intermediate. Here we report that cold and heat unfolding of Pfk-2 resembles the N2↔2I step of chemically induced unfolding. Moreover, cold unfolding appears to be as cooperative as that induced chemically and even more so than its heat-unfolding counterpart. Because Pfk-2 is a large homodimer of 66 kDa with a complex topology consisting of well-defined domains, these results are somewhat unexpected considering that cold unfolding has been described as a special kind of perturbation that decouples the cooperative unfolding of several proteins

Evolution, Metabolism and Molecular Mechanisms Underlying Extreme Adaptation of Euryarchaeota and Its Biotechnological Potential

Archaeal organisms harbor many unique genotypic and phenotypic properties, testifying their peculiar evolutionary status. Thus, the so‐called extremophiles must be adequately adapted to cope with many extreme environments with regard to metabolic processes, biological functions, genomes, and transcriptomes to overcome the challenges of life. This chapter will illustrate recent progress in the research on extremophiles from the phylum Euryarchaeota and compile their evolutive history, metabolic strategies, lipid composition, the structural adaptations of their enzymes to temperature, salinity, and pH and their biotechnological applications. Archaeal organisms have evolved to deal with one or more extreme conditions, and over the evolution, they have accumulated changes in order to optimize protein structure and enzyme activity. The structural basis of these adaptations resulted in the construction of a vast repertoire of macromolecules with particular features not found in other organisms. This repertoire can be explored as an inexhaustible source of biological molecules for industrial or biotechnological applications. We hope that the information compiled herein will open new research lines that will shed light on various aspects of these extremophilic microorganisms. In addition, this information will be a valuable resource for future studies looking for archaeal enzymes with particular properties

Historia evolutiva de la actividad piridoxal quinasa en el grupo de las hidroximetil pirimidina quinasas de bacterias

81 p.Las enzimas son las encargadas de catalizar un gran número de reacciones fundamentales a nivel celular. Desde un punto de vista evolutivo, las enzimas que provienen un ancestro común se agrupan en familias y super-familias. Dentro de una familia sus miembros comparten una alta similitud estructural, especialmente en el sitio activo. La plasticidad del plegamiento permite a la enzima reaccionar con diversos sustratos formando de esta manera enzimas promiscuas, una característica altamente ligada a la evolución de proteínas. La familia de quinasas de coenzimas que pertenece a la super-familia de riboquinasas, presenta grupos de enzimas que son específicas para un sustrato, como la enzima de Salmonella typhimurium (PDB: 1JXH) que tiene por función fosforilar hidroximetil pirimidina (HMP) y la enzima de E. coli, derivada del gen pdxY (PDB: 1TD2), que fosforila piridoxal (PL). Se han descrito además dos variantes bifuncionales con la capacidad de fosforilar tanto HMP como PL, formando por tanto una nueva subclase de enzima bifuncionales denominadas enzimas HMPK/PLK. Estas enzimas se encuentran en los organismos Bacillus subtilis (PDB: 2I5B) y Staphylococcus aureus (PDB: 4C5N) y están relacionadas filogenéticamente con las enzimas HMPKs. Mediante resurrección de enzimas ancestrales y estudios de cinética enzimática se demostró que el último ancestro en común de enzimas HMPK y HMPK/PLK (AncC1) presenta una promiscuidad por sustrato, siendo capaz de catalizar reacciones con PL y HMP. Sin embargo, la concentración de piridoxal necesaria para la catálisis está fuera del rango de las concentraciones fisiológicas y por lo tanto este ancestro sería específico por HMP. Análisis in silico de la proteína ancestral indican que existe una glutamina en el sitio activo de AncC1, la que está presente en las enzimas específicas por HMP y que no se encuentra en las enzimas bifuncionales de Bacillus subtilis y Staphylococcus aureus. En las variantes bifuncionales esta glutamina es reemplazada por metionina en el sitio activo. Basados en estos antecedentes, se propuso que el último ancestro en común del grupo de enzimas bifuncionales HMPK/PLK es bifuncional y que el cambio desde el ancestro específico AncC1 hacia las enzimas bifuncionales es debido a la mutación de una glutamina por una metionina en el sitio activo de estas enzimas.Para verificar esta hipótesis se realizó la inferencia de las secuencias ancestrales. Utilizando una filogenia actualizada se infirió la secuencia del último ancestro en común de las enzimas bifuncionales HMPK/PLK (AncBi) y la del último ancestro en común entre HMPK/PLK y HMPK (AncC2) utilizando los métodos estadísticos de máxima verosimilitud (ML) y método bayesiano (BA). Se generaron modelos por homología para los ancestros inferidos y se evaluaron las posibles interacciones con los sustratos de PL y HMP mediante docking. Además, se realizó una resurrección de la proteína ancestral AncBi/BA para una posterior caracterización cinética y mutación sitio dirigida para construcción de la mutante Q45M de AncC1. Al analizar el sitio activo y los residuos que se encontraban a 5 Å del ligando, se observa que el del AncC2 es semejante al de las enzimas específicas, ya que conserva el residuo de glutamina (Gln45) presente en las enzimas HMPKs específicas de Salmonella typhimurium y de Thermus thermophilus, el cual es reemplazado por una metionina en las enzimas bifuncionales de Bacillus subtilis y Staphylococcus aureus y en el ancestro AncBi. Esto sugiere que la enzima ancestral AncC2 es específica por HMP y que AncBi es bifuncional con actividad HMPK/PLK. Las curvas de saturación para los sustratos PL y HMP mostraron que la enzima AncBi posee una constante de Michaelis (Km) de 0.7 y 0.85 mM, respectivamente, mientras que los valores de eficiencia catalítica (kcat/Km) son de 73 M-1s-1 para PL y de 31 M-1s-1 para HMP, siendo ambos valores muy semejantes por lo que AncBi es una enzima bifuncional. Estos resultados nos hacen proponer que la actividad bifuncional es una novedad evolutiva y que la especificidad es el carácter ancestral de la familia, donde la actividad PLK habría surgido de manera convergente y de manera independiente. La mutación Q45M de AncC1 generó un aumento 21 veces la afinidad por PL con respecto a la enzima AncC1 silvestre. Sin embargo, no se observó un cambio significativo en la especificad por sustrato, debido principalmente a una disminución en el valor de kcat. De este modo, la mutante AncC1 Q45M se puede catalogar como un estado intermedio entre el ancestro AncC1 y el ancestro AncBi, haciendo falta otras mutaciones para lograr eficiencias catalíticas equivalentes a AncBi. Adicionalmente, se determinó la estructura del ancestro AncC1 mediante cristalografía y difracción de rayos X, y se analizó la conservación estructural con respecto a las enzimas actuales./ABSTRACT:Enzymes are responsible for catalyzing many fundamental cellular reactions. From an evolutionary point of view, enzymes that come from a common ancestor are grouped into families and super-families. Family members share high structural similarity, especially at the active site. Folding plasticity allows that enzymes react with various substrates, thereby forming promiscuous enzymes, a feature that is highly linked to the evolution of proteins. Kinase family of coenzymes belonging to the riboquinase superfamily, has groups of enzymes that are specific for a substrate such as the enzyme from Salmonella typhimurium (PDB: 1JXH) whose function is to phosphorylate hydroxymethyl pyrimidine (HMP) and the enzyme from E. coli codified in the pdxY gene (PDB: 1TD2) that phosphorylates pyridoxal (PL). Moreover, two bifunctional variants with the ability to phosphorylate HMP and PL have been described. These bifunctional enzymes form a new subclass called HMPK/PLK enzyme which are present in Bacillus subtilis (PDB: 2I5B) and Staphylococcus aureus (PDB: 4C5N). These bifunctional enzymes are phylogenetically related to the HMPKs enzymes. By ancestral enzyme reconstruction and kinetic studies we demonstrated that the last common ancestor of HMPK and HMPK/PLK (AncC1) was promiscuous, being able of catalyzing reactions with PL and HMP as substrates. However, pyridoxal concentration of needed for catalysis is far beyond the physiological range, and there for this ancestor as specific for HMP. In silico analysis of the ancestral protein AncC1 indicate the presence of a glutamine at the active site which is present in the HMP specific enzymes, but is absent in the bifunctional enzymes from Bacillus subtilis and Staphylococcus aureus. In the bifunctional enzymes this glutamine is replaced by methionine at the active site. Based on this background, it was proposed that the last common ancestor of the bifunctional HMPK/PLK enzymes group is bifunctional and that the change from the specific ancestor AncC1 to the bifunctional enzyme is due to a mutation of glutamine by a methionine at the active site of these enzymes. To verify this hypothesis inference of ancestral sequences was performed. The last common ancestor of bifunctional enzymes HMPK/PLK (AncBi) and the last common ancestor between HMPK/PLK and HMPK (AncC2) were inferred from an updated phylogeny, using statistical methods such as maximum likelihood (ML) and Bayesian method (BA). Homology models were generated for the inferred ancestors and interactions with HMP by PL substrates were assessed by docking. The resurrected AncBi ancestor was characterized kinetically and the AncC1 Q45M mutant was performed by site directed mutagenesis. Analysis of the active site and residues within 5 Å of the ligand shows that the active site of AncC2 is similar to the ones present in the specific enzymes since it retains the glutamine residue (Gln45) present in the HMPKs specific enzymes from Salmonella typhimurium and Thermus thermophilus. In the bifunctional enzymes from Bacillus subtilis, Staphylococcus aureus and in the AncBi ancestor, this residue is replaced by methionine. This suggests that the ancestral AncC2 enzyme is specific by HMP and that the ancestral AncBi have HMPK/PLK bifunctional activity. The saturation curves of the AncBi enzyme for PL and HMP substrates showed a Michaelis constant (Km) of 0.7 mM and 0.85 mM respectively, while the values of catalytic efficiency (kcat /Km) are 73 M-1s-1 to PL 31 M-1s-1 for HMP, being both values very similar so the AncBi is a bifunctional enzyme. These results lead us to propose that the bifunctional activity is the evolutionary novelty, and that specificity is the ancestral character of the family, where the PLK activity arises in a convergent and independent manner. The Q45M mutation of AncC1 generated a 21 times increase in the PL affinity with respect to the enzyme wild type AncC1. However a significant change in substrate specificity was not observed mainly due to a decrease in the kcat value. Thus, the AncC1 Q45M mutant can be classified as an intermediate state between the AncC1 ancestor and the AncBi ancestor, while another mutations will be need to achieve catalytic efficiencies equivalent to AncBi. Additionally, the structure of the AncC1 ancestor was determined by crystallography and X-ray diffraction, and the structural conservation regarding the current enzymes present was analyzed

Historia evolutiva de la actividad bifuncional hidroximetil pirimidina quinasa / piridoxal quinasa de la familia de quinasas de vitaminas dependientes de ATP

82 p.Las enzimas desempeñan una gran cantidad de funciones a nivel celular, catalizando reacciones mayormente de carácter específico por algún sustrato. En base a la función que desempeñan y su plegamiento, las enzimas se clasifican en familias y súper familias. En este trabajo estudiamos la familia de quinasas de vitaminas dependientes de ATP, la cual pertenece a la súper familia riboquinasa.La familia de quinasas de vitaminas presenta grupos de enzimas que son especificas por un sustrato, como la enzima de Salmonella typhimurium (PDB:1JXH) que tiene por función fosforilar hidroximetil pirimidina (HMP) y la enzima de E. coli derivada del gen pdxY (PDB:1TD2) que fosforila piridoxal (PL). Interesantemente en la familia de quinasas de vitaminas se encuentra caracterizada una enzima bifuncional codificada por el gen thiD de Bacillus subtilis (PDB:2I5B) que es capaz de fosforilar de manera inespecífica hidroximetil pirimidina y piridoxal. En términos de evolución de enzimas, hay dos mecanismos por los cuales las enzimas pueden evolucionar hacia una nueva especificidad; de una forma drástica perdiendo la función anterior y adquiriendo una nueva, o coexistiendo las dos actividades (actividad ancestral y nueva) durante una fase de transición.Evolutivamente no se tiene conocimiento si la actividad bifuncional de hidroximetil pirimidina quinasa (HMPK) / piridoxal quinasa (PLK) es un estado ancestral o es un rasgo más reciente en la historia evolutiva. En este trabajo propusimos que la actividad bifuncional es de carácter ancestral y que la familia evolucionó hacia la especificidad por hidroximetil pirimidina. Para corroborar esto se reconstruyó por métodos de inferencia filogenética la secuencia ancestral del grupo de las HMPK/PLK bifuncionales, la secuencia ancestral del grupo de las HMPK específicas y la secuencia del último ancestro en común para ambos grupos. Se generaron modelos por homologías para dichos ancestros y se evaluaron las posibles interacciones con los diferentes sustratos mediante docking. Al analizar el sitio activo y los residuos que se encontraban a 5 Å del ligando, se pudo inferir que el último ancestro en común para ambos grupos es semejante a las enzimas específicas, ya que conserva un residuo de glutamina (Gln44), presente en la mayoría de las 2 enzimas específicas por hidroximetil pirimidina, el cual es reemplazado por metionina en la enzima bifuncional de B. subtilis (Met44), lo que sugiere que la enzima ancestral es preferente por HMP por sobre PL. Experimentalmente se sintetizó el gen del último ancestro en común entre el grupo de las HMPK específicas y HMPK/PLK bifuncionales, con el fin de evaluar su capacidad de fosforilar ambos sustratos, PL y HMP. Se realizaron curvas de pH para conocer el intervalo en el cual la enzima presentaba la mayor actividad, correspondiente a pH 6 - 6,5 cuando se usó PL como sustrato, mientras que para HMP, ésta se encontró entre pH 7,5 - 8. Se realizó una curva de saturación de Mg- ATP, para encontrar la concentración saturante de dicho complejo para la enzima ancestral, la cual está sobre los 5 mM, encontrándose además inhibición a concentraciones por sobre 10 mM. Las curvas de saturación para los sustratos PL y HMP mostraron que la enzima posee una constante de Michaelis (Km) de 28 y 7 mM respectivamente, mientras que los valores de eficiencia catalítica ( kcat / Km ) para PL y HMP son de 7 M-1s-1 y de 57 M-1s-1 respectivamente, teniendo una preferencia por HMP 8 veces superior a la de PL. Estos valores experimentales confirman la suposición del análisis bioinformático de que la enzima es más preferente por HMP que por PL. Estos resultados nos hacen proponer que la actividad bifuncional es una novedad evolutiva, y que la especificidad es el carácter ancestral de la familia. La capacidad de fosforilar ambos sustratos con eficiencias catalíticas similares pudo haber surgido tardíamente en la familia por un evento de evolución paralela, ya que la actividad HMPK es el carácter ancestral./ABSTRACT: Enzymes play a lot of functions at the cellular level, catalyzing reactions mostly specific for a particular substrate. Based on their role and their fold, enzymes are classified into families and superfamilies. In this paper we study the family of ATP dependent vitamin kinases, which belongs to the ribokinase superfamily. The family of vitamin kinases has groups of enzymes that are specific for a substrate, such as Salmonella typhimurium (PDB: 1JXH) whose function is to phosphorylate hydroxymethyl pyrimidine (HMP) and the enzyme derived from pdxY gene of E. coli (PDB: 1TD2) that phosphorylates pyridoxal (PL). Interestingly, a bifunctional enzyme from Bacillus subtilis encoded by gene thiD has been described (PDB: 2I5B), that is capable of non-specifically phosphorylate hydroxymethyl pyrimidine and pyridoxal. In terms of enzyme evolution, there are two mechanisms by which enzymes can evolve into a new specificity; by dramatically losing a previous function and acquiring a new one, or by coexistence of two activities (ancestral and new activities) during a transition phase.Evolutionarily it is unknown whether the bifunctional hydroxymethyl pyrimidine kinase (HMPK) / pyridoxal kinase (PLK) activity is an ancestral trait or a more recent event in evolutionary history. In this work we proposed that the bifunctional activity is an ancestral trait and thus the family evolved towards hydroxymethyl pyrimidine specificity. To corroborate this, we reconstructed the ancestral sequence of the bifunctional group of HMPK / PLK, the ancestral sequence of the specific group of HMPK specific and the sequence of the last common ancestor for both groups by means of phylogenetic inference methods. Homology models were generated for these ancestors and possible interactions with its different substrates were evaluated by docking. Upon analysis of the active site and the residues at 5 Å of the ligand, it was possible to infer that the last common ancestor for both groups is similar to the specific enzymes as it retains a glutamine residue (Gln44) present in most hydroxymethyl pyrimidine specific enzymes, which is replaced by a methionine in the bifunctional enzyme from B. subtilis (Met44), suggesting that the ancestral enzyme prefers HMP over PL. Experimentally, the gene of the last common ancestor between the group of specific HMPK and dual HMPK / PLK was synthesized in order to evaluate the ability to phosphorylate both substrates, PL and HMP. pH curves were performed to determine the interval where the enzyme had the highest activity, corresponding to pH 6 - 6.5 when PL was used as a substrate, whereas for HMP, this was found between pH 7.5 - 8. We performed a saturation curve of Mg-ATP, to find the saturation concentration of this complex for the ancestral enzyme, which is about 5 mM, followed by further inhibition at concentrations above 10 mM. Saturation curves for PL and HMP substrates showed that the enzyme has a Michaelis constant (Km) of 28 mM and 7 respectively, while the catalytic efficiency (kcat / Km) values for PL and HMP are 7 M- 1s-1 and 57 M-1s-1 respectively, thus having a preference 8 times higher for HMP over PL. These experimental values confirmed the bioinformatic analysis assumption that the enzyme prefers HMP rather than PL. These results lead us to propose that the bifunctional activity is an evolutionary novelty, and that specificity is the ancestral character of the family. The ability to phosphorylate both substrates with similar catalytic efficiencies could have arisen later in the family from an event of parallel evolution, as HMPK activity is the ancestral character

Chemical modification of SH groups of E. coli Phosphofructokinase-2 induces subunit dissociation: Monomers are inactive but preserve ligand binding properties

Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with N- (1-pyrenil)maleimide results in an enzyme form that is inactive. However, the rate of modification is drastically reduced in the presence of the allosteric effector MgATP. The stoichiometry of the label incorporation was found to be 2.03 ± 0.035 mol of the reagent/mol of subunit, in agreement with the number of titratable SH groups by 5,5'-dithiobis(2-nitrobenzoic acid) in the labeled protein. HPLC gel filtration experiments demonstrate that native Pfk-2 is a dimer in the absence of ligands, while in the presence of MgATP a dimer- tetramer transition is promoted. In contrast, the modified enzyme eluted as a monomer and the presence of MgATP was not able to induce aggregation. Although the modified monomers are inactive, the dissociation constants for the substrates and the allosteric effector MgATP, measured by following the fluorescence of the binding probe, are the same as for the native enzyme. Quenching of pyrene fluorescence emission of labeled phosphofructokinase-2 monomers by acrylamide gave downward curved Stern–Volmer plots, with very similar quenching efficiencies for the control and for the fruc- tose-6-P and MgATP– enzyme complexes. These results show the presence of SH groups in the interface of Pfk-2 subunits, critical for subunit interactions, and that con- formational changes occurring through the dimers are essential for catalytic activity

Specificity evolution of the ADP-dependent sugar kinase family - in silico studies of the glucokinase/phosphofructokinase bifunctional enzyme from Methanocaldococcus jannaschii

In several archaea of the Euryarchaeota, the glycolytic flux proceeds through a modified version of the Embden-Meyerhof pathway, where the phosphofructokinase and glucokinase enzymes use ADP as the phosphoryl donor. These enzymes are homologous to each other. In the hyperthermophilic methanogenic archaeon Methanocaldococcus jannaschii, it has been possible to identify only one homolog for these enzymes, which shows both ADP-dependent glucokinase and phosphofructokinase activity. This enzyme has been proposed as an ancestral form in this family. In this work we studied the evolution of this protein family using the Bayesian method of phylogenetic inference and real value evolutionary trace in order to test the ancestral character of the bifunctional enzyme. Additionally, to search for specificity determinants of these two functions, we have modeled the bifunctional protein and its interactions with both sugar substrates using protein-ligand docking and restricted molecular dynamics. Th

Influence of ligands on the aggregation of the normal and mutant forms of phosphofructokinase 2 of Escherichia coli

The aggregation states of Escherichia coli phosphofructokinase 2 (Pfk-2) and of a mutant enzyme (Pfk-2*) altered in the inhibitory allosteric site for MgATP were measured in the presence and in the absence of substrates and products of the reaction. When sucrose gradient ultracentrifugation experiments were performed in the absence of added ligands, both enzymes sedimented as dimers. Likewise, at low concentrations of both substrates (0.1 mm) the aggregation state of Pfk-2 and Pfk-2* corresponded to a dimer. However, in the presence of 1 mm MgATP alone, Pfk-2 sedimented as a tetramer, whereas Pfk-2* sedimented as a dimer. At a low fructose 6-phosphate concentration (0.1 mm) and an inhibitory concentration of MgATP (4 mm), Pfk-2 sedimented as a tetramer. However, at the same MgATP concentration but at a higher fructose-6-P concentration (1 mm), a condition under which Pfk-2 is not inhibited by the Mg-nucleotide complex, the enzyme sedimented as a dimer. Pfk-2* is not inhibited under th