Insights into the molecular relationships between malate and lactate dehydrogenases: structural and biochemical properties of monomeric and dimeric intermediates of a mutant of tetrameric L-[LDH-like] malate dehydrogenase from the halophilic archaeon Haloarcula marismortui

L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes. LDHs are tetramers, whereas MalDHs can be either dimeric or tetrameric. To gain insight into molecular relationships between LDHs and MalDHs, we studied folding intermediates of a mutant of the LDH-like MalDH (a protein with LDH-like structure and MalDH enzymatic activity) from the halophilic archaeon Haloarcula marismortui (Hm MalDH). Crystallographic analysis of Hm MalDH had shown a tetramer made up of two dimers interacting mainly via complex salt bridge clusters. In the R207S/R292S Hm MalDH mutant, these salt bridges are disrupted. Its structural parameters, determined by neutron scattering and analytical centrifugation under different conditions, showed the protein to be a tetramer in 4 M NaCl. At lower salt concentrations, stable oligomeric intermediates could be trapped at a given pH, temperature, or NaCl solvent concentration. The spectroscopic properties and enzymatic behavior of monomeric, dimeric, and tetrameric species were thus characterized. The properties of the dimeric intermediate were compared to those of dimeric intermediates of LDH and dimeric MalDHs. A detailed analysis of the putative dimer-dimer contact regions in these enzymes provided an explanation of why some can form tetramers and others cannot. The study presented here makes Hm MalDH the best characterized example so far of an LDH-like MalDH.     

Molecular adaptation: the malate dehydrogenase from the extreme halophilic bacterium Salinibacter ruber behaves like a non-halophilic protein

Malate dehydrogenase from the extreme halophilic bacterium, Salinibacter ruber (Sr MalDH) was purified and characterised as a tetramer by sedimentation velocity measurements, showing the enzyme belongs to the LDH-like group of MalDHs. In contrast to most other halophilic enzymes, which unfold when incubated at low salt concentration, Sr MalDH is completely stable in absence of salt. Its amino acid composition does not display the strong acidic character specific of halophilic proteins. The enzyme displays a strong KCl-concentration dependent variation in K(m) for oxaloacetate, but not for the NADH co-factor. Its activity is reduced by high salt concentration, but remains sufficient for the enzyme to sustain catalysis at approximately 30% of its maximal rates in 3 M KCl. The properties of the protein were compared with those from other LDH-like MalDHs of bacterial and archaeal origins, showing that Sr MalDH in fact behaves like a non-halophilic enzyme.     

Evolution of Cryptosporidium parvum lactate dehydrogenase from malate dehydrogenase by a very recent event of gene duplication

We have expressed the L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (malDH) genes from the apicomplexan Cryptosporidium parvum (CpLDH1 and CpMalDH1) as maltose-binding protein (MBP) fusion proteins in Escherichia coli. The substrate specificities, enzymatic kinetics, and oligomeric states of these two parasite enzymes have been characterized. By taking advantage of recently completed and ongoing apicomplexan genome sequencing projects, we identified additional MalDH genes from Plasmodium spp., Toxoplasma gondii, and Eimeria tenella that were previously unavailable. All apicomplexan MalDHs appeared to be cytosolic and no organellar homologs were identified from the completely sequenced P. falciparum genome and other ongoing apicomplexan genome-sequencing projects. Using these expanded apicomplexan LDH and MalDH sequence databases, we reexamined their phylogenetic relationships and reconfirmed their relationship to alpha-proteobacterial MalDHs. All LDH and MalDH enzymes from apicomplexans were monophyletic within the LDH-like MalDH group (i.e., MalDH resembling LDH) as a sister to alpha-proteobacterial MalDHs. All apicomplexan LDHs, with the exception of CpLDH1, formed a separate clade from their MalDH counterparts, indicating that these LDHs were evolved from an ancestral apicomplexan MalDH by a gene duplication coupled with functional conversion before the expansion of apicomplexans. Finally, CpLDH1 was consistently placed together with CpMalDH1 within the apicomplexan MalDH cluster, confirming an early working hypothesis that CpLDH1 was probably evolved from the same ancestor of CpMalDH1 by a very recent gene duplication that occurred after C. parvum diverged from other apicomplexans.     

Halophilic adaptation: novel solvent protein interactions observed in the 2.9 and 2.6 A resolution structures of the wild type and a mutant of malate dehydrogenase from Haloarcula marismortui

Previous biophysical studies of tetrameric malate dehydrogenase from the halophilic archaeon Haloarcula marismortui (Hm MalDH) have revealed the importance of protein-solvent interactions for its adaptation to molar salt conditions that strongly affect protein solubility, stability, and activity, in general. The structures of the E267R stability mutant of apo (-NADH) Hm MalDH determined to 2.6 A resolution and of apo (-NADH) wild type Hm MalDH determined to 2.9 A resolution, presented here, highlight a variety of novel protein-solvent features involved in halophilic adaptation. The tetramer appears to be stabilized by ordered water molecule networks and intersubunit complex salt bridges "locked" in by bound solvent chloride and sodium ions. The E267R mutation points into a central ordered water cavity, disrupting protein-solvent interactions. The analysis of the crystal structures showed that halophilic adaptation is not aimed uniquely at "protecting" the enzyme from the extreme salt conditions, as may have been expected, but, on the contrary, consists of mechanisms that harness the high ionic concentration in the environment.     

Porphyromonas gingivalis: an invasive and evasive opportunistic oral pathogen

Archaea that live at high salt concentrations are a phylogenetically diverse group of microorganisms. They include the heterotrophic haloarchaea (class Halobacteria) and some methanogenic Archaea, and they inhabit both oxic and anoxic environments. In spite of their common hypersaline environment, halophilic archaea are surprisingly diverse in their nutritional demands, range of carbon sources degraded (including hydrocarbons and aromatic compounds) and metabolic pathways. The recent discovery of a new group of extremely halophilic Euryarchaeota, the yet uncultured Nanohaloarchaea, shows that the archaeal diversity and metabolic variability in hypersaline environments is higher than hitherto estimated.     

Effects of nicotine on the biosynthesis of carotenoids in halophilic Archaea (class Halobacteria): an HPLC and Raman spectroscopy study

Extremophiles - Nicotine has a profound influence on the carotenoid metabolism in halophilic Archaea of the class Halobacteria. In a study of Halobacterium salinarum, Haloarcula marismortui and...     

Fructans of the saline world

Saline and hypersaline environments make up the largest ecosystem on earth and the organisms living in such water-restricted environments have developed unique ways to cope with high salinity. As such these organisms not only carry significant industrial potential in a world where freshwater supplies are rapidly diminishing, but they also shed light upon the origins and extremes of life. One largely overlooked and potentially important feature of many salt-loving organisms is their ability to produce fructans, fructose polymers widely found in various mesophilic Eubacteria and plants, with potential functions as storage carbohydrates, aiding stress tolerance, and acting as virulence factors or signaling molecules. Intriguingly, within the whole archaeal domain of life, Archaea possessing putative fructan biosynthetic enzymes were found to belong to the extremely halophilic class of Halobacteria only, indicating a strong, yet unexplored link between the fructan syndrome and salinity. In fact, this link may indeed lead to novel strategies in fighting the global salinization problem. Hence this review explores the unknown world of fructanogenic salt-loving organisms, where water scarcity is the main stress factor for life. Within this scope, prokaryotes and plants of the saline world are discussed in detail, with special emphasis on their salt adaptation mechanisms, the potential roles of fructans and fructosyltransferase enzymes in adaptation and survival as well as future aspects for all fructanogenic salt-loving domains of life.     

Halocin SH10 production by an extreme haloarchaeon Natrinema sp. BTSH10 isolated from salt pans of South India

Halobacteria, members of the domain Archaea that live under extremely halophilic conditions, are often considered as dependable source for deriving novel enzymes, novel genes, bioactive compounds and other industrially important molecules. Protein antibiotics have potential for application as preserving agents in food industry, leather industry and in control of infectious bacteria. Halocins are proteinaceous antibiotics synthesized and released into the environment by extreme halophiles, a universal characteristic of halophilic bacteria. Herein, we report the production of halocin (SH10) by an extremely halophilic archeon Natrinema sp. BTSH10 isolated from salt pan of Kanyakumari, Tamilnadu, India and optimization of medium for enhanced production of halocin. It was found that the optimal conditions for maximal halocin production were 42 °C, pH 8.0, and 104 h of incubation at 200 rpm with 2% (V/V) inoculum concentration in Zobell’s medium containing 3 M NaCl, Galactose, beef extract, and calcium chloride as additional supplements. Results indicated scope for fermentation production of halocin for probable applications using halophilic archeon Natrinema sp. BTSH10.     

嗜盐古细菌的系统发育分析

用“Clustalw”和“PHYLIP”程序包分析嗜盐古细菌16S rRNA序列,建立了嗜盐古细菌的系统发育树。比较分析的结果进一步支持了以前的结论,即嗜盐古细菌在自然系统分类上应被分成嗜盐菌科的6个属。此系统发育分析方法不仅体现了在嗜盐古细菌属一级分类上的优势,而且还可能被用作一种相应于以《伯杰氏细菌系统分类手册》(第三卷)为基础的嗜盐古细菌种一级分类上的代换方法。实际上,这种系统发育分析方法比其他建立在表型特性基础上的分类系统更真实地反映了嗜盐古细菌内的亲缘关系。该方法的其他运算细节在本文中也进行了讨论。     

The 2.9A resolution crystal structure of malate dehydrogenase from Archaeoglobus fulgidus: mechanisms of oligomerisation and thermal stabilisation

The crystal structure of malate dehydrogenase from the hyperthermophilic archaeon Archeoglobus fulgidus, in complex with its cofactor NAD, was solved at 2.9A resolution. The crystal structure shows a compact homodimer with one coenzyme bound per subunit. The substrate binding site is occupied by a sulphate ion. In order to gain insight into adaptation mechanisms, which allow the protein to be stable and active at high temperatures, the 3D structure was compared to those of several thermostable and hyperthermostable homologues, and to halophilic malate dehydrogenase. The hyperthermostable A. fulgidus MalDH protein displays a reduction of the solvent-exposed surface, an optimised compact hydrophobic core, a high number of hydrogen bonds, and includes a large number of ion pairs at the protein surface. These features occur concomitantly with a reduced number of residues in the protein subunit, due to several deletions in loop regions. The loops are further stiffened by ion pair links with secondary structure elements. A. fulgidus malate dehydrogenase is the only dimeric protein known to date that belongs to the [LDH-like] MalDH family. All the other known members of this family are homo-tetramers. The crystal structures revealed that the association of the dimers to form tetramers is prevented by several deletions, taking place at the level of two loops that are known to be essential for the tetramerisation process within the LDH and [LDH-like] MalDH enzymes.     

Adaptation to environmental temperature is a major determinant of molecular evolutionary rates in archaea

Methods to infer the ancestral conditions of life are commonly based on geological and paleontological analyses. Recently, several studies used genome sequences to gain information about past ecological conditions taking advantage of the property that the G+C and amino acid contents of bacterial and archaeal ribosomal DNA genes and proteins, respectively, are strongly influenced by the environmental temperature. The adaptation to optimal growth temperature (OGT) since the Last Universal Common Ancestor (LUCA) over the universal tree of life was examined, and it was concluded that LUCA was likely to have been a mesophilic organism and that a parallel adaptation to high temperature occurred independently along the two lineages leading to the ancestors of Bacteria on one side and of Archaea and Eukarya on the other side. Here, we focus on Archaea to gain a precise view of the adaptation to OGT over time in this domain. It has been often proposed on the basis of indirect evidence that the last archaeal common ancestor was a hyperthermophilic organism. Moreover, many results showed the influence of environmental temperature on the evolutionary dynamics of archaeal genomes: Thermophilic organisms generally display lower evolutionary rates than mesophiles. However, to our knowledge, no study tried to explain the differences of evolutionary rates for the entire archaeal domain and to investigate the evolution of substitution rates over time. A comprehensive archaeal phylogeny and a non homogeneous model of the molecular evolutionary process allowed us to estimate ancestral base and amino acid compositions and OGTs at each internal node of the archaeal phylogenetic tree. The last archaeal common ancestor is predicted to have been hyperthermophilic and adaptations to cooler environments can be observed for extant mesophilic species. Furthermore, mesophilic species present both long branches and high variation of nucleotide and amino acid compositions since the last archaeal common ancestor. The increase of substitution rates observed in mesophilic lineages along all their branches can be interpreted as an ongoing adaptation to colder temperatures and to new metabolisms. We conclude that environmental temperature is a major factor that governs evolutionary rates in Archaea.     

Cloning,expression, and efficient purification in Escherichia coli of a halophilic nucleoside diphosphate kinase from the moderate halophile Halomonas sp. #593

Most typical halophilic enzymes from extremely halophilic archaea require high concentrations of salt for their activity and stability. These enzymes are inactive in Escherichia coli unless refolded in the presence of salts in vitro. In this report, we describe cloning of the ndk gene of nucleoside diphosphate kinase from a moderately halophilic eubacterium and overexpression of the protein in E. coli as an N-terminal hexa-His fusion to facilitate its purification on Ni-NTA affinity resin. We demonstrate evidence that the protein is properly folded and exhibits the same specific activity and stability as the native protein from Halomonas cells.     

The evolution and evolutionary consequences of marginal thermostability in proteins

When we seek to explain the characteristics of living systems in their evolutionary context, we are often interested in understanding how and why certain properties arose through evolution, and how these properties then affected the continuing evolutionary process. This endeavor has been assisted by the use of simple computational models that have properties characteristic of natural living systems but allow simulations over evolutionary timescales with full transparency. We examine a model of the evolution of a gene under selective pressure to code for a protein that exists in a prespecified folded state at a given growth temperature. We observe the emergence of proteins with modest stabilities far below those possible with the model, with a denaturation temperature tracking the simulation temperature, despite the absence of selective pressure for such marginal stability. This demonstrates that neither observations of marginally stable proteins, nor even instances where increased stability interferes with function, provide evidence that marginal stability is an adaptation. Instead the marginal stability is the result of a balance between predominantly destabilizing mutations and selection that shifts depending on effective population size. Even if marginal stability is not an adaptation, the natural tendency of proteins toward marginal stability, and the range of stabilities that occur during evolution, may have significant effect on the evolutionary process.     

Halophilic adaptation of enzymes

It is now clear that the understanding of halophilic adaptation at a molecular level requires a strategy of complementary experiments, combining molecular biology, biochemistry, and cellular approaches with physical chemistry and thermodynamics. In this review, after a discussion of the definition and composition of halophilic enzymes, the effects of salt on their activity, solubility, and stability are reviewed. We then describe how thermodynamic observations, such as parameters pertaining to solvent–protein interactions or enzyme-unfolding kinetics, depend strongly on solvent composition and reveal the important role played by water and ion binding to halophilic proteins. The three high-resolution crystal structures now available for halophilic proteins are analyzed in terms of haloadaptation, and finally cellular response to salt stress is discussed briefly. Received: July 11, 1999 / Accepted: December 27, 1999     

From the salt marsh to the ID-card: Bacteriorhodopsin as a functional material in nanobiotechnology

Bacteriorhodopsin (BR) is an evolutionary highly optimized photochromic retinal protein, which is found in extremely halophilic bacteria, e.g., in salt marshes. We demonstrated that starting from the wildtype as a blueprint by means of gene technology and biotechnology a versatile material for optical information recording can be developed. BR is structurally related to the visual pigment rhodopsin. It is the key molecule in the halobacterial photosynthetic system — an alternative to the chlorophyll-dependent photosynthesis. Its biological function ist that of a light-driven proton pump. In the halobacterial cell — which are found e.g. in salt marshes — it converts light energy into chemical energy, i.e. a proton gradient over the cell membrane, which finally supplies ATP to the cell. The photochromic properties of BR are very attractive compared to those of known organic photochromic compounds, in particular as far as longevity under exposure to oxygen and light is concerned. This is one of the reasons why we try to utilized this evolutionary optimized biomaterial for technical applications in particular in optical data storage and processing. As the biological function of BR is optimized for energy conversion, the physical properties of BR need to be tuned to turn this molecule into a material which matches the requirements of optical applications in data storage and processing. Gene technology is a powerful tool for the controlled modification of physical properties of a biomolecule like BR. In technical applications water needs to be omitted. However, the function of biomaterials strictly depends on the presence of water. Membrane proteins are much less dependent on the presence of water which makes them good candidates for technical applications. We showed that BR can be processed into dry polymeric films where its function is preserved. In a field test where ID-cards comprising BR-based inks as security elements it has been demonstrated that biomaterials may be integrated in active form as functional components into conventional technical applications. Conventional nanomaterials supply properties to a product, biomaterials supply functions.     

Evolutionary divergence and salinity-mediated selection in halophilic archaea.

Halophilic (literally salt-loving) archaea are a highly evolved group of organisms that are uniquely able to survive in and exploit hypersaline environments. In this review, we examine the potential interplay between fluctuations in environmental salinity and the primary sequence and tertiary structure of halophilic proteins. The proteins of halophilic archaea are highly adapted and magnificently engineered to function in an intracellular milieu that is in ionic balance with an external environment containing between 2 and 5 M inorganic salt. To understand the nature of halophilic adaptation and to visualize this interplay, the sequences of genes encoding the L11, L1, L10, and L12 proteins of the large ribosome subunit and Mn/Fe superoxide dismutase proteins from three genera of halophilic archaea have been aligned and analyzed for the presence of synonymous and nonsynonymous nucleotide substitutions. Compared to homologous eubacterial genes, these halophilic genes exhibit an inordinately high proportion of nonsynonymous nucleotide substitutions that result in amino acid replacement in the encoded proteins. More than one-third of the replacements involve acidic amino acid residues. We suggest that fluctuations in environmental salinity provide the driving force for fixation of the excessive number of nonsynonymous substitutions. Tinkering with the number, location, and arrangement of acidic and other amino acid residues influences the fitness (i.e., hydrophobicity, surface hydration, and structural stability) of the halophilic protein. Tinkering is also evident at halophilic protein positions monomorphic or polymorphic for serine; more than one-third of these positions use both the TCN and the AGY serine codons, indicating that there have been multiple nonsynonymous substitutions at these positions. Our model suggests that fluctuating environmental salinity prevents optimization of fitness for many halophilic proteins and helps to explain the unusual evolutionary divergence of their encoding genes.     

Crowding in extremophiles: linkage between solvation and weak protein-protein interactions, stability and dynamics, provides insight into molecular adaptation

The study of the molecular adaptation of microorganisms to extreme environments (solvent, temperature, etc.) has provided tools to investigate the complex relationships between protein-solvent and protein-protein interactions, protein stability and protein dynamics, and how they are modulated by the crowded environment of the cell. We have evaluated protein-solvent and protein-protein interactions by solution experiments (analytical ultracentrifugation, small angle neutron and X-ray scattering, density) and crystallography, and protein dynamics by energy resolved neutron scattering. This review concerns work from our laboratory on (i) proteins from extreme halophilic Archaea, and (ii) psychrophile, mesophile, thermophile and hyperthermophile bacterial cells.