Michael J. Danson

Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium.

BACKGROUND The structural basis of adaptation of enzymes to low temperature is poorly understood. Dimeric citrate synthase has been used as a model enzyme to study the structural basis of thermostability, the structure of the enzyme from organisms living in habitats at 55 degrees C and 100 degrees C having previously been determined. Here the study is extended to include a citrate synthase from an Antarctic bacterium, allowing us to explore the structural basis of cold activity and thermostability across the whole temperature range over which life is known to exit. RESULTS We report here the first crystal structure of a cold-active enzyme, citrate synthase, isolated from an Antarctic bacterium, at a resolution of 2.09 A. In comparison with the same enzyme from a hyperthermophilic host, the cold-active enzyme has a much more accessible active site, an unusual electrostatic potential distribution and an increased relative flexibility of the small domain compared to the large domain. Several other features of the cold-active enzyme were also identified: reduced subunit interface interactions with no intersubunit ion-pair networks; loops of increased length carrying more charge and fewer proline residues; an increase in solvent-exposed hydrophobic residues; and an increase in intramolecular ion pairs. CONCLUSIONS Enzymes from organisms living at the temperature extremes of life need to avoid hot or cold denaturation yet maintain sufficient structural integrity to allow catalytic efficiency. For hyperthermophiles, thermal denaturation of the citrate synthase dimer appears to be resisted by complex networks of ion pairs at the dimer interface, a feature common to other hyperthermophilic proteins. For the cold-active citrate synthase, cold denaturation appears to be resisted by an increase in intramolecular ion pairs compared to the hyperthermophilic enzyme. Catalytic efficiency of the cold-active enzyme appears to be achieved by a more accessible active site and by an increase in the relative flexibility of the small domain compared to the large domain.

The crystal structure of citrate synthase from the thermophilic Archaeon, Thermoplasma acidophilum

BACKGROUND The Archaea constitute a phylogenetically distinct, evolutionary domain and comprise organisms that live under environmental extremes of temperature, salinity and/or anaerobicity. Different members of the thermophilic Archaea tolerate temperatures in the range 55-110 degrees C, and the comparison of the structures of their enzymes with the structurally homogolous enzymes of mesophilic organisms (optimum growth temperature range 15-45 degrees C) may provide important information on the structural basis of protein thermostability. We have chosen citrate synthase, the first enzyme of the citric acid cycle, as a model enzyme for such studies. RESULTS We have determined the crystal structure of Thermoplasma acidophilum citrate synthase to 2.5 A and have compared it with the citrate synthase from pig heart, with which it shares a high degree of structural homology, but little sequence identity (20%). CONCLUSIONS The three-dimensional structural comparison of thermophilic and mesophilic citrate synthases has permitted catalytic and substrate-binding residues to be tentatively assigned in the archaeal, thermophilic enzyme, and has identified structural features that may be responsible for its thermostability.

Metabolic Pathway Promiscuity in the Archaeon Sulfolobus solfataricus Revealed by Studies on Glucose Dehydrogenase and 2-Keto-3-deoxygluconate Aldolase

The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between d-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.

The Structural Basis of Protein Halophilicity

Abstract The halophilic Archaea live in hypersaline environments and maintain an osmotic balance by accumulating intracellular concentrations of salt (mainly KCl) that are isotonic with the exterior. Therefore, their cellular components are adapted to concentrations of KCl approaching 5 M and often require these levels of salt for stability and function. Here, we consider the effects of hypersalinity on protein structure and then review the known structures of proteins from the halophilic Archaea in the context of these effects. Specific proteins considered are ferredoxin, malate dehydrogenase, dihydrofolate reductase, dihydrolipoamide dehydrogenase and elongation factor Tu. From the available data, models for the structural basis of halophilicity are discussed and analysed.

Archaebacteria: the comparative enzymology of their central metabolic pathways.

Publisher Summary It is noted that for the purposes of establishing the phylogenetic status of the archaebacteria, much emphasis has been placed on the molecular biology of these organisms and on the chemical nature of their cell walls and membranes. However, it is also becoming clear that the pathways of metabolism in archaebacteria and their constituent enzymes are equally fruitful areas for investigation. This chapter focuses on the enzymology of archaebacteria, not in isolation but in comparison with that of eubacteria and eukaryotes. The enzymes of the central metabolic pathways have been reviewed because these pathways are thought to be some of the first cellularly established metabolic routes and are the most studied and well-characterized systems in non-archaebacterial species. Archaebacteria grow in extreme environments and therefore their macro- molecules will be structurally adapted to function under such conditions. Thermoacidophilic archaebacteria grow at temperatures between 55 and 110°C and in pH values as low as pH 1-2. Although there can be no temperature differential between the outside and inside of a cell, it is thought that cytoplasmic pH values are approximately neutral.

Structure, function and stability of enzymes from the Archaea

The Archaea include microorganisms growing in some of the most extreme environments on earth. Consequently, their cellular components are remarkably stable entities and have considerable potential in the biotechnology industry. Here, we review the structure of archaeal enzymes in the context of their ability to function at extremes of temperature, salinity, pH and pressure.

The dependence of enzyme activity on temperature: determination and validation of parameters

Traditionally, the dependence of enzyme activity on temperature has been described by a model consisting of two processes: the catalytic reaction defined by DeltaG(Dagger)(cat), and irreversible inactivation defined by DeltaG(Dagger)(inact). However, such a model does not account for the observed temperature-dependent behaviour of enzymes, and a new model has been developed and validated. This model (the Equilibrium Model) describes a new mechanism by which enzymes lose activity at high temperatures, by including an inactive form of the enzyme (E(inact)) that is in reversible equilibrium with the active form (E(act)); it is the inactive form that undergoes irreversible thermal inactivation to the thermally denatured state. This equilibrium is described by an equilibrium constant whose temperature-dependence is characterized in terms of the enthalpy of the equilibrium, DeltaH(eq), and a new thermal parameter, T(eq), which is the temperature at which the concentrations of E(act) and E(inact) are equal; T(eq) may therefore be regarded as the thermal equivalent of K(m). Characterization of an enzyme with respect to its temperature-dependent behaviour must therefore include a determination of these intrinsic properties. The Equilibrium Model has major implications for enzymology, biotechnology and understanding the evolution of enzymes. The present study presents a new direct data-fitting method based on fitting progress curves directly to the Equilibrium Model, and assesses the robustness of this procedure and the effect of assay data on the accurate determination of T(eq) and its associated parameters. It also describes simpler experimental methods for their determination than have been previously available, including those required for the application of the Equilibrium Model to non-ideal enzyme reactions.

Metabolism of glucose via a modified Entner-Doudoroff pathway in the thermoacidophilic archaebacterium Thermoplasma acidophilum

It has been found that the thermoacidophilic archaebacterium, Thermoplasma acidophilum, can metabolise glucose via a modified Entner‐Doudoroff pathway involving non‐phosphorylated intermediates. Pyruvate and glyceraldehyde are the first products, the glyceraldehyde then being further metabolised to a second molecule of pyruvate via 2‐phosphoglycerate. Intermediates of this pathway have been identified by enzymic analysis or by thin‐layer chromatography and the individual enzymes involved have been assayed and their kinetic parameters determined. Comparisons are made with the pathways of glucose metabolism in other archaebacteria. p]Archaebacteria (Thermoplasma) Entner‐Doudoroff pathway Glucose metabolism

The structural basis for substrate promiscuity in 2-keto-3-deoxygluconate aldolase from the Entner-Doudoroff pathway in Sulfolobus solfataricus.

The hyperthermophilic Archaea Sulfolobus solfataricus grows optimally above 80 °C and metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to d-2-keto-3-deoxygluconate (KDG). KDG aldolase (KDGA) then catalyzes the cleavage of KDG to d-glyceraldehyde and pyruvate. It has recently been shown that all the enzymes of this pathway exhibit a catalytic promiscuity that also enables them to be used for the metabolism of galactose. This phenomenon, known as metabolic pathway promiscuity, depends crucially on the ability of KDGA to cleave KDG and d-2-keto-3-deoxygalactonate (KDGal), in both cases producing pyruvate and d-glyceraldehyde. In turn, the aldolase exhibits a remarkable lack of stereoselectivity in the condensation reaction of pyruvate and d-glyceraldehyde, forming a mixture of KDG and KDGal. We now report the structure of KDGA, determined by multiwavelength anomalous diffraction phasing, and confirm that it is a member of the tetrameric N-acetylneuraminate lyase superfamily of Schiff base-forming aldolases. Furthermore, by soaking crystals of the aldolase at more than 80 °C below its temperature activity optimum, we have been able to trap Schiff base complexes of the natural substrates pyruvate, KDG, KDGal, and pyruvate plus d-glyceraldehyde, which have allowed rationalization of the structural basis of promiscuous substrate recognition and catalysis. It is proposed that the active site of the enzyme is rigid to keep its thermostability but incorporates extra functionality to be promiscuous.

A new understanding of how temperature affects the catalytic activity of enzymes

The two established thermal properties of enzymes are their activation energy and their thermal stability, but experimental data do not match the expectations of these two properties. The recently proposed Equilibrium Model (EM) provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. It was formulated as a mathematical model, and fits the known experimental data. Importantly, the EM gives rise to a number of new insights into the molecular basis of the temperature control of enzymes and their environmental adaptation and evolution, it is consistent with active site properties, and it has fundamental implications for enzyme engineering and other areas of biotechnology.