A Rigidifying Salt-Bridge Favors the Activity of Thermophilic Enzyme at High Temperatures at the Expense of Low-Temperature Activity

Although enzymes from thermophiles thriving in hot habitats are more stable than their mesophilic homologs, they are often less active at low temperatures. One theory suggests that extra stabilizing interactions found in thermophilic enzymes may increase their rigidity and decrease enzymatic activity at lower temperatures. We used acylphosphatase as a model to study how flexibility affects enzymatic activity. This enzyme has a unique structural feature in that an invariant arginine residue, which takes part in catalysis, is restrained by a salt-bridge in the thermophilic homologs but not in its mesophilic homologs. Here, we demonstrate the trade-offs between flexibility and enzymatic activity by disrupting the salt-bridge in a thermophilic acylphosphatase and introducing it in the mesophilic human homolog. Our results suggest that the salt-bridge is a structural adaptation for thermophilic acylphosphatases as it entropically favors enzymatic activity at high temperatures by restricting the flexibility of the active-site residue. However, at low temperatures the salt-bridge reduces the enzymatic activity because of a steeper temperature-dependency of activity

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

Role of Proteome Physical Chemistry in Cell Behavior.

We review how major cell behaviors, such as bacterial growth laws, are derived from the physical chemistry of the cell's proteins. On one hand, cell actions depend on the individual biological functionalities of their many genes and proteins. On the other hand, the common physics among proteins can be as important as the unique biology that distinguishes them. For example, bacterial growth rates depend strongly on temperature. This dependence can be explained by the folding stabilities across a cell's proteome. Such modeling explains how thermophilic and mesophilic organisms differ, and how oxidative damage of highly charged proteins can lead to unfolding and aggregation in aging cells. Cells have characteristic time scales. For example, E. coli can duplicate as fast as 2-3 times per hour. These time scales can be explained by protein dynamics (the rates of synthesis and degradation, folding, and diffusional transport). It rationalizes how bacterial growth is slowed down by added salt. In the same way that the behaviors of inanimate materials can be expressed in terms of the statistical distributions of atoms and molecules, some cell behaviors can be expressed in terms of distributions of protein properties, giving insights into the microscopic basis of growth laws in simple cells

Characterization of the Allosteric Properties of Thermus thermophilus Phosphofructokinase and the Sources of Strong Inhibitor Binding Affinity and Weak Inhibitory Response

Characterization of allosteric properties of phosphofructokinase from the extreme thermophile Thermus thermophilus (TtPFK) using thermodynamic linkage analysis revealed several peculiarities. Inhibition and activation of Fru-6-P binding by the allosteric effectors phosphoenolpyruvate (PEP) and MgADP are entropically-driven in TtPFK. It is also curious that PEP binding affinity is unusually strong in TtPFK when compared to PFKs from Escherichia coli, Bacillus stearothermophilus, and Lactobacillus delbrueckii, while the magnitude of the allosteric inhibition by PEP is much smaller in TtPFK. In an effort to understand the source of weak inhibition, a putative network of residues between the allosteric site and the nearest active site was identified from the three-dimensional structures of BsPFK. Three of the residues in this network, D59, T158, and H215, are not conserved in TtPFK, and, due to their nature (N59, A158, S215), are unlikely to be involved in the same non-covalent interactions seen in BsPFK. The triple chimeric substitution N59D/A158T/S215H, results in a 2.5 kcal mol-1 increase in the coupling free energy, suggesting that the region containing these residues may be important for propagation of inhibitory response. The individual substitutions at each position resulted in an increase in the coupling free energy, and the double substitutions displayed additivity of these changes. The chimeric substitution made at N59 suggests that the polar nature of the asparagine at position 59 is key for the enhanced binding of PEP. The non-conserved R55 was found to be particularly important for the enhanced binding of PEP in TtPFK, as chimeric substitutions R55G and R55E resulted in a 3.5 kcal mol-1 and 4.5 kcal mol-1 decrease in the binding affinity for PEP, respectively. Our results also confirm the observations previously made in PFKs from E. coli and B. stearothermophilus, that the ability of the effector to bind is independent of its ability to produce allosteric response. We show that several substitutions result in a decrease in binding affinity of PEP to TtPFK, while dramatically enhancing its ability to inhibit (N59D, R55G, R55E). Similarly, some substitutions, like S215H and A158T show an enhanced inhibition by PEP, while having no effect on its binding affinity

Insight into the multicopper oxidases stability

Dissertation presented to obtain the PhD degree in BiochemistryThis dissertation portrays recent development on the knowledge of the stability determinants and of functional characteristics of multicopper oxidases (MCO). Multicopper oxidases are a family of enzymes that includes laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2), ascorbate oxidase (L-ascorbate oxygen oxidoreductase, EC 1.10.3.3) and ceruloplasmin (Fe2+ oxygen oxidoreductase, EC 1.16.3.1). MCO are characterized by having four copper ions that are classified into three distinct types of copper sites, namely type 1 (T1), type 2 (T2) and type 3 (T3). The classical T1 copper site comprises two histidine residues and a cysteine residue arranged in a distorted trigonal geometry around the copper ion with bonding distances approx. 2.0 Å (1 Å=0.1 nm); a weaker fourth methionine ligand completes the tetrahedral geometry. The copper–cysteine linkage is characterized by an intense S(π)→Cu(dx2−y2) CT (charge transfer) absorption band at approximately 600 nm, and a narrow parallel hyperfine splitting A\\ = (43–90)×10−4 cm−1 in the electron paramagnetic resonance (EPR) spectrum. The function of the T1 copper site is to shuttle electrons from substrates to the trinuclear copper centre where molecular oxygen is reduced to two molecules of water during the complete four-electron catalytic cycle. The trinuclear center contains a T2 copper coordinated by two histidine residues and one water molecule, lacks strong absorption bands and exhibits a large parallel hyperfine splitting in the EPR spectrum (A\\ = (150–201)×10−4 cm−1). The T2 copper site is in close proximity to two T3 copper ions, which are each coordinated by three histidine residues and typically coupled, for example, through a dioxygen molecule. The T3 or coupled binuclear copper site is characterized by an intense absorption band at 330 nm originating from the bridging ligand and by the absence of an EPR signal due to the antiferromagnetically coupling of the copper ions.(...)Apoio financeiro da FCT e do FSE no âmbito do Quadro Comunitário de Apoio, BD nº SFRH/BD/31444/200

Síntesis de oligosacáridos fucosilados en medio convencional y no-convencional empleando la α-L-fucosidasa de Thermotoga maritima

Human milk is the ideal food during the first months in the new-born life with his composition adapted to their nutritional needs. Fucosylated oligosaccharides are found in human milk and have diverse beneficial effects on the health of infants. In this work, the effect of pH, temperature and water activity (aw) on both the hydrolytic and transglycosylation activity on the synthesis of fucosylated oligosaccharides was evaluated by using the α-L-fucosidase from Thermotoga maritima. The fucosidase show an optimum pH range of 6-8 for hydrolytic activity and 7-10 for transglycosylation activity. In addition, when temperature was raised, an increase on both the hydrolytic and transglycosylation activities was observed, obtaining the highest enzymatic activity at 95 ° C. Additionally, the highest productivity in the synthesis of fucosylated oligosaccharides (3.54 mM/h) was obtained at pH 8 and 95 ° C by using pNP-fucose (3.5 mM) as donor substrate and lactose (438 mM) as acceptor substrate. Subsequently, the effect of aw was evaluated; aw was decreased by the addition of dimethyl sulfoxide (DMSO), acetone and acetonitrile. In the reaction medium with acetone (aw 0.97, 0.95 0.93) and acetonitrile (aw 0.96, 0.93 and 0.91), a hydrolytic activity greater than 100% was obtained with respect to the control (water media), while with DMSO at aw DMSO> acetonitrile (0.51> 0.42> 0.18 mM/h). With the addition of DMSO and acetone, transglycosylation activity was favoured over the hydrolytic one, this by obtaining a transglycosylation/hydrolysis rate (rS/H) of 1.21 and 1.43, respectively; on the contrary, in the medium with acetonitrile, the hydrolytic activity prevailed (rS/H 0.59), however, all solvents allowed to obtain a higher rS/H compared to the control (0.39). Additionally, the effect of temperature on the structure of the α-L-fucosidase of T. maritima was evaluated by molecular dynamics simulation (DM). In the DM at 60-90 °C, no significant changes were observed in the average RMSD (RMSDavg)values, indicating the stability of the enzyme in that range of temperatures. However, at 95 °C there was a significant increase on both, the RMSDavg and average RMSF (RMSFavg) values, which reflect important conformational changes in the enzyme structure. Furthermore, results indicate that temperature contributes to changes in the secondary structure of the enzyme, causing a decrease in the content of α-helices with the subsequent increased in irregular conformation, which would explain the loss of enzymatic activity at high temperatures (200 °C). Likewise, the temperature influences the secondary structure of the active site of the enzyme, which can adopt different conformations: open, intermediate or closed. Finally, with the molecular docking technique, differences were observed in the interaction of α-L-fucosidase with the ligand L-fucose and pNP-Fuc, as the temperature increased a lower free binding energy and higher affinity of the enzyme for the corresponding substrate was observed.La leche humana es el alimento ideal en los primeros meses de vida del infante y su composición está adaptada a sus necesidades nutricionales. En la leche humana se encuentran oligosacáridos fucosilados, los cuales tienen diversos efectos benéficos en la salud de los infantes. Debido a las ventajas anteriormente mencionas, el objetivo de este trabajo fue aumentar el rendimiento de síntesis de oligosacáridos fucosilados, evaluando los parámetros de pH, temperatura y actividad de agua (aw) en la actividad hidrolítica y de transglicosilación de la α-L-fucosidasa de Thermotoga maritima. La fucosidasa presentó un intervalo de pH óptimo de 6-8 para la actividad hidrolítica y de 7-10 para la actividad de transglicosilación. Además, al aumentar la temperatura se observó un incremento tanto en la actividad hidrolítica como de transglicosilación, obteniéndose la mayor actividad enzimática a 95 °C. Asimismo, la productividad más alta en la síntesis de oligosacáridos fucosilados (3.54 mM/h) se registró a pH 8 y 95 °C empleando pNP-fucosa (3.5 mM) como sustrato donador y lactosa (438 mM) como sustrato aceptor. Posteriormente se evalúo el efecto de la aw, la cual se disminuyó mediante la adición de dimetil sulfóxido (DMSO), acetona y acetonitrilo. En el medio de reacción con acetona (aw 0.97, 0.95 0.93) y acetonitrilo (aw 0.96, 0.93 y 0.91) se obtuvo una actividad hidrolítica mayor al 100% respecto al ensayo control (medio acuoso), mientras que con DMSO a awDMSO >acetonitrilo (0.51>0.42>0.18 mM/h). La adición de acetona y DMSO favoreció la actividad de transglicosilación sobre la actividad hidrolítica, esto se comprobó al obtenerse una tasa de transglicosilación/hidrólisis (rS/H) de 1.21 y 1.43, respectivamente. Por el contrario, en el medio con acetonitrilo la actividad hidrolítica prevalece (rS/H 0.59), no obstante, con todos los solventes se obtuvo una mayor rS/H respecto al ensayo control (0.39). Adicionalmente, se evalúo el efecto de la temperatura en la estructura de la α-L-fucosidasa de T. maritima mediante simulación de dinámica molecular (DM). En las DM realizadas a 60-90 °C no se observaron cambios significativos en los valores del promedio de RMSD (RMSDprom) indicando la estabilidad de la enzima en ese intervalo de temperaturas, sin embargo, a 95 °C se registró un incremento significativo en el RMSDprom y promedio de RMSF (RMSFprom) reflejando cambios conformacionales importantes en la estructura de la enzima. Además, los resultados obtenidos indican que la temperatura contribuye a los cambios en la estructura secundaria de la enzima, ocasionando una disminución en el contenido de α-hélices con el subsecuente aumento de conformación irregular, lo que explicaría la pérdida de actividad enzimática a temperaturas elevadas (200 °C). Asimismo, la temperatura influye en la estructura secundaria del sitio activo de la enzima, el cual puede adoptar diferentes conformaciones: abierta, intermedia o cerrada. Finalmente, con la técnica de acoplamiento molecular se observaron diferencias en la interacción de la α-L-fucosidasa con los ligandos L-fucosa y pNP-Fuc, reflejándose en una menor energía libre de unión y una mayor afinidad de la enzima por el sustrato conforme se incrementó la temperatura

Kinetic stability and temperature adaptation. Observations from a cold adapted subtilisin-like serine protease.

Life on earth is found everywhere where water is found, meaning that life has adapted to extremely varied environments. Thus, protein structures must adapt to a myriad of environmental stressors while maintaining their functional forms. In the case of enzymes, temperature is one of the main evolutionary pressures, affecting both the stability of the structure and the rate of catalysis. One of the solutions Nature has come up with to maintain activity and stability in harsh environments over biological relevant timescales, are kinetically stable proteins. This thesis will outline work carried out on the kinetically stable VPR, a cold active subtilisin-like serine protease and discuss our current understanding of protein kinetic stability, temperature adaptation and our current hypothesis of the molecular interactions contributing to the stability of VPR. The research model that we have used to study these attributes consists of the cold active VPR and its thermostable structural homolog AQUI. The results discussed in this thesis will be on the importance of calcium, the role of prolines in loops, the role of a conserved N-terminal tryptophan residue and lastly primary observations on differences in active site dynamics between VPR and AQUI. A model is proposed of a native structure that unfolds in a highly cooperative manner. This cooperativity can be disrupted, however, by modifying calcium binding of the protein or via mutations that affect how the N-terminus interacts with the rest of the protein. The N-terminus likely acts as a kinetic lock that infers stability to the rest of the structure through many different interactions. Some of these interactions may be strengthened via proline residues, that seemingly act as anchor points that tend to maintain correct orientation between these parts of the protein as thermal energy is increased in the system. Our results give a deeper insight into the nature of the kinetic stability, the importance of cooperativity during unfolding of kinetically stable proteases, synergy between distant parts of the protein through proline mutations and how different calcium binding sites have vastly differing roles. The results provide a solid ground for continuing work in designing enzyme variants with desired stabilities and activities and improve our understanding of kinetically stable systems.The Icelandic Research Fund [grant number 162977-051