The Experts below are selected from a list of 1194 Experts worldwide ranked by ideXlab platform
Evelyn Arndt - One of the best experts on this subject based on the ideXlab platform.
-
the alpha operon equivalent genome region in the extreme halophilic archaebacterium haloarcula halobacterium marismortui
Journal of Biological Chemistry, 1992Co-Authors: Thomas Scholzen, Evelyn ArndtAbstract:Abstract The genome region of the extreme halophilic archaebacterium Haloarcula marismortui equivalent to the alpha-operon of Escherichia coli has been characterized. In H. marismortui, the alpha-operon was found to be located immediately upstream from the S9 gene cluster. The gene order in the Halobacterial alpha-operon, given according to the gene products, is tRNA(Ser), HmaS13, HmaS4, HmaS11, and HmaRp alpha. Compared to the corresponding operon from E. coli, the Halobacterial gene organization differs in (i) the presence of a gene for tRNA(Ser) (GCU), (ii) the reversed order of the genes for the ribosomal proteins HmaS11 and HmaS4, and (iii) the absence of the gene coding for the ribosomal protein L17. The primary structure of HmaRp alpha shows high similarity to a subunit of eukaryotic RNA polymerase II (YeaRpB3, HsaRpB33), whereas the similarity to the eubacterial alpha-subunit of RNA polymerase is only weak.
-
the genes for ribosomal protein l15 and the protein equivalent to secy in the archaebacterium haloarcula halobacterium marismortui
Biochimica et Biophysica Acta, 1992Co-Authors: Evelyn ArndtAbstract:The nucleotide sequences of the L15 gene and the secY gene, which form the last two genes of the S10/spc-operon region of Haloarcula marismortui, have been determined. The HmaL15 protein sequence translated from the DNA is 164 amino acids long, revealing 10 amino acids more at the C-terminus than the published protein sequence. The deduced HmasecY protein sequence has 487 amino acids and shows significant homology to its counterparts in Methanococcus vannielii and Escherichia coli. The gene order of the Halobacterial gene cluster is similar to that in the methanogens and in eubacteria.
Dimitry Y. Sorokin - One of the best experts on this subject based on the ideXlab platform.
-
Table_1_New Insights Into the Polar Lipid Composition of Extremely Halo(alkali)philic Euryarchaea From Hypersaline Lakes.docx
2019Co-Authors: Nicole J. Bale, Dimitry Y. Sorokin, Ellen C. Hopmans, Michel Koenen, Irene W. C. Rijpstra, Laura Villanueva, Hans Wienk, Jaap Sinninghe S. DamstéAbstract:We analyzed the polar membrane lipids of 13 strains of halo(alkali)philic euryarchaea from hypersaline lakes. Nine belong to the class Halobacteria, representing two functional groups: aerobic polysaccharide utilizers and sulfur-respiring anaerobes. The other four strains represent halo(alkali)philic methanogens from the class Methanomicrobia and a recently discovered class Methanonatronarchaeia. A wide range of polar lipids were detected across the 13 strains including dialkyl glycerol diethers (archaeols), membrane-spanning glycerol tetraethers and diether-based cardiolipins. The archaeols contained a range of core lipid structures, including combinations of C20 and C25 isoprenoidal alkyl chains, unsaturations, and hydroxy moieties. Several diether lipids were novel, including: (a) a phosphatidylglycerolhexose (PG-Gly) headgroup, (b) a N,N,N-trimethyl aminopentanetetrol (APT)-like lipid with a methoxy group in place of a hydroxy group on the pentanetetrol, (c) a series of polar lipids with a headgroup with elemental composition of either C12H25NO13S or C12H25NO16S2, and (d) novel cardiolipins containing a putative phosphatidylglycerolphosphate glycerophosphate (PGPGP) polar moiety. We found that the lipid distribution of the 13 strains could be generally separated into two groups, the methanogens (group) and the Halobacteria (class) based on the presence of specific core lipids. Within the methanogens, adaption to a high or more moderate salt concentration resulted in different ratios of glycerol dialkyl glycerol tetraethers (GDGTs) to archaeol. The methanogen Methanosalsum natronophilum AME2T had the most complex diether lipid composition of any of the 13 strains, including hydroxy archaeol and macrocyclic archaeol which we surmise is an order-specific membrane adaption. The zwitterionic headgroups APT and APT-Me were detected only in the Methanomicrobiales member Methanocalculus alkaliphilus AMF2T which also contained the highest level of unsaturated lipids. Only alkaliphilic members of the Natrialbales order contained PGPGP cardiolipins and the PG-Gly headgroup. The four analyzed neutrophilic members of the Halobacteria were characterized by the presence of sulfur-containing headgroups and glycolipids. The presence of cardiolipins with one or more i-C25 alkyl chains, generally termed extended archaeol (EXT-AR), in one of the Methanonatronarchaeia strains was unexpected as only one other order of methanogenic archaea has been reported to produce EXT-AR. We examined this further by looking into the genomic potential of various archaea to produce EXT-AR.
-
New Insights Into the Polar Lipid Composition of Extremely Halo(alkali)philic Euryarchaea From Hypersaline Lakes
Frontiers Media S.A., 2019Co-Authors: Nicole J. Bale, Dimitry Y. Sorokin, Ellen C. Hopmans, Michel Koenen, Irene W. C. Rijpstra, Laura Villanueva, Hans Wienk, Jaap Sinninghe S. DamstéAbstract:We analyzed the polar membrane lipids of 13 strains of halo(alkali)philic euryarchaea from hypersaline lakes. Nine belong to the class Halobacteria, representing two functional groups: aerobic polysaccharide utilizers and sulfur-respiring anaerobes. The other four strains represent halo(alkali)philic methanogens from the class Methanomicrobia and a recently discovered class Methanonatronarchaeia. A wide range of polar lipids were detected across the 13 strains including dialkyl glycerol diethers (archaeols), membrane-spanning glycerol tetraethers and diether-based cardiolipins. The archaeols contained a range of core lipid structures, including combinations of C20 and C25 isoprenoidal alkyl chains, unsaturations, and hydroxy moieties. Several diether lipids were novel, including: (a) a phosphatidylglycerolhexose (PG-Gly) headgroup, (b) a N,N,N-trimethyl aminopentanetetrol (APT)-like lipid with a methoxy group in place of a hydroxy group on the pentanetetrol, (c) a series of polar lipids with a headgroup with elemental composition of either C12H25NO13S or C12H25NO16S2, and (d) novel cardiolipins containing a putative phosphatidylglycerolphosphate glycerophosphate (PGPGP) polar moiety. We found that the lipid distribution of the 13 strains could be generally separated into two groups, the methanogens (group) and the Halobacteria (class) based on the presence of specific core lipids. Within the methanogens, adaption to a high or more moderate salt concentration resulted in different ratios of glycerol dialkyl glycerol tetraethers (GDGTs) to archaeol. The methanogen Methanosalsum natronophilum AME2T had the most complex diether lipid composition of any of the 13 strains, including hydroxy archaeol and macrocyclic archaeol which we surmise is an order-specific membrane adaption. The zwitterionic headgroups APT and APT-Me were detected only in the Methanomicrobiales member Methanocalculus alkaliphilus AMF2T which also contained the highest level of unsaturated lipids. Only alkaliphilic members of the Natrialbales order contained PGPGP cardiolipins and the PG-Gly headgroup. The four analyzed neutrophilic members of the Halobacteria were characterized by the presence of sulfur-containing headgroups and glycolipids. The presence of cardiolipins with one or more i-C25 alkyl chains, generally termed extended archaeol (EXT-AR), in one of the Methanonatronarchaeia strains was unexpected as only one other order of methanogenic archaea has been reported to produce EXT-AR. We examined this further by looking into the genomic potential of various archaea to produce EXT-AR
-
proteomic analysis of methanonatronarchaeum thermophilum amet1 a representative of a putative new class of euryarchaeota methanonatronarchaeia
Genes, 2018Co-Authors: Manuel Ferrer, Sergio Ciordia, Maria Carmen Mena, Rafael Bargiela, Dimitry Y. Sorokin, Yuri I Wolf, Kira S MakarovaAbstract:The recently discovered Methanonatronarchaeia are extremely halophilic and moderately thermophilic methyl-reducing methanogens representing a novel class-level lineage in the phylum Euryarchaeota related to the class Halobacteria. Here we present a detailed analysis of 1D-nano liquid chromatography–electrospray ionization tandem mass spectrometry data obtained for “Methanonatronarchaeum thermophilum” AMET1 grown in different physiological conditions, including variation of the growth temperature and substrates. Analysis of these data allows us to refine the current understanding of the key biosynthetic pathways of this triple extremophilic methanogenic euryarchaeon and identify proteins that are likely to be involved in its response to growth condition changes.
-
proteomic analysis of methanonatronarchaeum thermophilum amet1 a representative of a putative new class of euryarchaeota methanonatronarchaeia
Genes, 2018Co-Authors: Manuel Ferrer, Sergio Ciordia, Maria Carmen Mena, Rafael Bargiela, Dimitry Y. Sorokin, Yuri I Wolf, Eugene V Koonin, Kira S MakarovaAbstract:The recently discovered Methanonatronarchaeia are extremely halophilic and moderately thermophilic methyl-reducing methanogens representing a novel class-level lineage in the phylum Euryarchaeota related to the class Halobacteria. Here we present a detailed analysis of 1D-nano liquid chromatography–electrospray ionization tandem mass spectrometry data obtained for “Methanonatronarchaeum thermophilum” AMET1 grown in different physiological conditions, including variation of the growth temperature and substrates. Analysis of these data allows us to refine the current understanding of the key biosynthetic pathways of this triple extremophilic methanogenic euryarchaeon and identify proteins that are likely to be involved in its response to growth condition changes.
-
discovery of extremely halophilic methyl reducing euryarchaea provides insights into the evolutionary origin of methanogenesis
Nature Reviews Microbiology, 2017Co-Authors: Sergio Ciordia, Maria Carmen Mena, Manuel Ferrer, Dimitry Y. Sorokin, Kira S Makarova, Ben Abbas, Peter N Golyshin, Erwin A Galinski, Alexander Y MerkelAbstract:Methanogenic archaea are major players in the global carbon cycle and in the biotechnology of anaerobic digestion. The phylum Euryarchaeota includes diverse groups of methanogens that are interspersed with non-methanogenic lineages. So far, methanogens inhabiting hypersaline environments have been identified only within the order Methanosarcinales. We report the discovery of a deep phylogenetic lineage of extremophilic methanogens in hypersaline lakes and present analysis of two nearly complete genomes from this group. Within the phylum Euryarchaeota, these isolates form a separate, class-level lineage 'Methanonatronarchaeia' that is most closely related to the class Halobacteria. Similar to the Halobacteria, 'Methanonatronarchaeia' are extremely halophilic and do not accumulate organic osmoprotectants. The high intracellular concentration of potassium implies that 'Methanonatronarchaeia' employ the 'salt-in' osmoprotection strategy. These methanogens are heterotrophic methyl-reducers that use C 1 -methylated compounds as electron acceptors and formate or hydrogen as electron donors. The genomes contain an incomplete and apparently inactivated set of genes encoding the upper branch of methyl group oxidation to CO2 as well as membrane-bound heterodisulfide reductase and cytochromes. These features differentiate 'Methanonatronarchaeia' from all known methyl-reducing methanogens. The discovery of extremely halophilic, methyl-reducing methanogens related to haloarchaea provides insights into the origin of methanogenesis and shows that the strategies employed by methanogens to thrive in salt-saturating conditions are not limited to the classical methylotrophic pathway.
Dieter Oesterhelt - One of the best experts on this subject based on the ideXlab platform.
-
orthorhombic two dimensional crystal form of purple membrane bacteriorhodopsin membrane protein structure Halobacteria crystallization
2016Co-Authors: Hartmut Michel, Dieter Oesterhelt, Richard HendersontAbstract:A new two-dimensional crystal form of purple membrane has been obtained in vitro. It is produced by the joint use of a cationic detergent, dodecyltrimethylammonium chlo- ride, and the nonionic detergent, Triton X-100. It primarily forms large, rolled-up sheets that look like needles in the light microscope. Liposomes and tubes are also observed. The ab- sorption maximum of the new form of purple membrane is blue-shifted by 6 nm and its density is slightly lower than the natural form of purple membrane. The new form of purple membrane is orthorhombic with space group p2212i and cell dimensions 57.6 X 73.5 A. Four molecules of bacteriorhodopsin occupy the unit cell with an area per molecule close to that found in the native p3 structure. The projected structure to 6.5-A resolution was determined by electron microscopy and dif- fraction. It shows an identical molecular structure to that of the p3 form and determines the position of the polypeptide boundary.
-
signal processing and flagellar motor switching during phototaxis of halobacterium salinarum
Genome Research, 2003Co-Authors: Torsten Nutsch, Dieter Oesterhelt, Wolfgang Marwan, Ernst Dieter GillesAbstract:Prokaryotic taxis, the active search of motile cells for the best environmental conditions, is one of the paradigms for signal transduction. The search algorithm implemented by the cellular biochemistry modulates the probability of switching the rotational direction of the flagellar motor, a nanomachine that propels prokaryotic cells. On the basis of the well-known biochemical mechanisms of chemotaxis in Escherichia coli, kinetic modeling of the events leading from chemoreceptor activation by ligand binding to the motility response has been performed with great success. In contrast to Escherichia coli, Halobacterium salinarum, in addition, responds to visible light, which is sensed through specific photoreceptors of different wavelength sensitivity (phototaxis). Light stimuli of defined intensity and time course can be controlled precisely, which facilitates input-output measurements used for system analysis of the molecular network connecting the sensory receptors to the flagellar motor switch. Here, we analyze the response of Halobacterial cells to single and double-pulse light stimuli and present the first kinetic model for prokaryotic cells that couples the signal-transduction pathway with the flagellar motor switch. Modeling based on experimental data supports the current biochemical model of Halobacterial phototaxis. Moreover, the simulations demonstrate that motor switching occurs through subsequent rate-limiting steps, which are both under sensory control, suggesting that two signals may be involved in Halobacterial phototaxis.
-
a novel mode of sensory transduction in archaea binding protein mediated chemotaxis towards osmoprotectants and amino acids
The EMBO Journal, 2002Co-Authors: Maia V Kokoeva, Kaiflorian Storch, Christian Klein, Dieter OesterheltAbstract:Directly upstream of the Halobacterium salinarum transducer genes basT and htpIV we identified two open reading frames (orfs) with significant homologies to genes encoding binding proteins for amino acids and compatible solutes, respectively. Behavioral testing of deletion mutants indicates that Halobacterial chemotaxis towards branched-chain amino acids as well as compatible osmolytes of the betaine family requires both a binding and a transducer protein. We therefore named the binding/transducer proteins BasB/BasT for branched-chain and sulfur-containing amino acids and CosB/CosT for compatible solutes. Our data support a signaling mechanism with the binding proteins functioning as lipid-anchored receptors interacting with the extracellular domain of their cognate transducers. Inspection of the Halobacterial genome suggests that BasB and CosB exclusively mediate chemotaxis responses without any additional role in transport, which is in contrast to bacterial binding proteins, which are always part of ABC transport systems. The CosB/CosT system is the first instance of a chemotaxis signaling pathway for organic osmolytes in the living world and natural abundance 13C-NMR analysis of cytoplasmic extracts suggests that H.salinarum utilizes these solutes for osmotic adaptation.
-
the fdx gene encoding the 2fe 2s ferredoxin of halobacterium salinarium h halobium
Molecular Genetics and Genomics, 1993Co-Authors: Felicitas Pfeifer, Johannes Griffig, Dieter OesterheltAbstract:The gene encoding the [2Fe--2S] ferredoxin (fdx gene) was isolated from Halobacterium salinarium using two oligonucleotides deduced from the ferredoxin sequence as probes. Cosmid DNAs exhibiting hybridization were isolated, the fdx gene was localized to smaller subfragments and the nucleotide sequence determined. The 390 bp coding sequence is located in the Halobacterial FI-DNA and transcribed as a 440 nucleotide mRNA. S1 mapping indicated that the 5' terminus of the mRNA maps immediately upstream of the ATG start codon. The promoter box A, centred around position -25 (5' AC-TATG 3'), and box B (TG) elements at the start of the transcript resemble the sequences of a typical archaeal promoter. The restriction pattern of an approximately 50 kb region surrounding the fdx gene is conserved in various Halobacterium species. The Halobacterial ferredoxin and the major gas vesicle protein GvpA exhibit up to 70% similarity to their respective counterparts in cyanobacteria suggesting lateral gene transfer between the organisms. These similarities prompted a more detailed investigation of the relative positions of the genes in the Halobacterial genome.
-
improved purification crystallization and primary structure of pyruvate ferredoxin oxidoreductase from halobacterium halobium
FEBS Journal, 1992Co-Authors: Wulf Plaga, Friedrich Lottspeich, Dieter OesterheltAbstract:An improved purification procedure, including nickel chelate affinity chromatography, is reported which resulted in a crystallizable pyruvate:ferredoxin oxidoreductase preparation from Halobacterium halobium. Crystals of the enzyme were obtained using potassium citrate as the precipitant. The genes coding for pyruvate:ferredoxin oxidoreductase were cloned and their nucleotide sequences determined. The genes of both subunits were adjacent to one another on the Halobacterial genome. The derived amino acid sequences were confirmed by partial primary structure analysis of the purified protein. The structural motif of thiamin-diphosphate-binding enzymes was unequivocally located in the deduced amino acid sequence of the small subunit.
Victor Castro-fernandez - One of the best experts on this subject based on the ideXlab platform.
-
Structural and Kinetic Insights Into the Molecular Basis of Salt Tolerance of the Short-Chain Glucose-6-Phosphate Dehydrogenase From Haloferax volcanii
'Frontiers Media SA', 2021Co-Authors: Nicolás Fuentes-ugarte, Victor Castro-fernandez, Sixto M. Herrera, Pablo Maturana, Victoria GuixéAbstract:Halophilic enzymes need high salt concentrations for activity and stability and are considered a promising source for biotechnological applications. The model study for haloadaptation has been proteins from the Halobacteria class of Archaea, where common structural characteristics have been found. However, the effect of salt on enzyme function and conformational dynamics has been much less explored. Here we report the structural and kinetic characteristics of glucose-6-phosphate dehydrogenase from Haloferax volcanii (HvG6PDH) belonging to the short-chain dehydrogenases/reductases (SDR) superfamily. The enzyme was expressed in Escherichia coli and successfully solubilized and refolded from inclusion bodies. The enzyme is active in the presence of several salts, though the maximum activity is achieved in the presence of KCl, mainly by an increment in the kcat value, that correlates with a diminution of its flexibility according to molecular dynamics simulations. The high KM for glucose-6-phosphate and its promiscuous activity for glucose restrict the use of HvG6PDH as an auxiliary enzyme for the determination of halophilic glucokinase activity. Phylogenetic analysis indicates that SDR-G6PDH enzymes are exclusively present in Halobacteria, with HvG6PDH being the only enzyme characterized. Homology modeling and molecular dynamics simulations of HvG6PDH identified a conserved NLTX2H motif involved in glucose-6-phosphate interaction at high salt concentrations, whose residues could be crucial for substrate specificity. Structural differences in its conformational dynamics, potentially related to the haloadaptation strategy, were also determined
-
Assessing the halophilic character of ADP-dependent sugar kinases from the archeon order Methanosarcinales
Proceedings of MOL2NET 2018 International Conference on Multidisciplinary Sciences 4th edition, 2019Co-Authors: Pablo Cea, Felipe Gonzalez-ordenes, Victor Castro-fernandezAbstract:Halophilic organisms have evolved to live in environments of high salinity, therefore theirmolecular machinery has adapted to carry out its functions in presence of molar concentrations ofsalt. Most of the work aimed to understand the structural adaptations of these proteins has beendone using proteins from the archeon class Halobacteria. Proteins from these organisms arecharacterized by a low abundance of basic residues and a high amount of acidic residues, whichaccumulate on the protein surface, coupled with a reduction of bulky hydrophobic residues in itscore [1]. Nevertheless, halophilic organisms have been reported in a wide variety of taxa, includingother archaea orders, which their adaptation mechanisms have not been explored. To evaluate theubiquity of the protein structural adaptations found in Halobacteria, we built homology modelsof ADP-dependent kinases from halophilic and non-halophilic organisms of the archaeal orderMethanosarcinales and compared them to models from Halobacterial and Eucariotic proteins.Our results show that proteins from halophilic organisms of the Methanosarcinales order do notshow the classical bias in amino acid composition observed in Halobacteria, like the reduction ofthe hydrophobic core and negative surface charge. However, experimental characterization of theADP-dependent phosphofructokinase of the halophilic organism Methanohalobium evestigatum(from Methanosarcinales order) confirmed that the protein is indeed halotolerant, and thischaracter can be further exacerbated in presence of osmolytes commonly found on halophilicarchaea, like betaine [2]. These results suggest that the adaptations required to maintain thestructure and function of a protein in extreme salt concentrations can vary widely betweendifferent organisms. These adaptations do not rely exclusively on the amino acidic composition,being instead a product of the coevolutionary process between the protein and its intracellularenvironment. Fondecyt 1150460 References [1] Graziano, G., a Merlino, A. (2014). Molecular bases of protein halotolerance. Biochimica et Biophysica Acta(BBA) - Proteins and Proteomics, 1844(4), 850–858[2] Sowers, K. R., a Gunsalus, R. P. (1995). Halotolerance in Methanosarcina spp.: Role of N (sup (epsilon))-Acetyl-(beta)-Lysine,(alpha)-Glutamate, Glycine Betaine, and K (sup+) as Compatible Solutes for OsmoticAdaptation. Applied and environmental microbiology, 61(12), 4382-4388.
-
ADP-Dependent Kinases From the Archaeal Order Methanosarcinales Adapt to Salt by a Non-canonical Evolutionarily Conserved Strategy.
Frontiers in Microbiology, 2018Co-Authors: Felipe Gonzalez-ordenes, Pablo Cea, Nicolás Fuentes-ugarte, Sebastián M. Muñoz, Ricardo A. Zamora, Diego Leonardo, Richard Charles Garratt, Victor Castro-fernandezAbstract:Halophilic organisms inhabit hypersaline environments where the extreme ionic conditions and osmotic pressure have driven the evolution of molecular adaptation mechanisms. Understanding such mechanisms is limited by the common difficulties encountered in cultivating such organisms. Within the Euryarchaeota, for example, only the Halobacteria and the order Methanosarcinales include readily cultivable halophilic species. Furthermore, only the former have been extensively studied in terms of their component proteins. Here, in order to redress this imbalance, we investigate the halophilic adaptation of glycolytic enzymes from the ADP-dependent phosphofructokinase/glucokinase family (ADP-PFK/GK) derived from organisms of the order Methanosarcinales. Structural analysis of proteins from non-halophilic and halophilic Methanosarcinales shows an almost identical composition and distribution of amino acids on both the surface and within the core. However, these differ from those observed in Halobacteria or Eukarya. Proteins from Methanosarcinales display a remarkable increase in surface lysine content and have no reduction to the hydrophobic core, contrary to the features ubiquitously observed in Halobacteria and which are thought to be the main features responsible for their halophilic properties. Biochemical characterization of recombinant ADP-PFK/GK from M. evestigatum (halophilic), and M. mazei (non-halophilic) shows the activity of both these extant enzymes to be only moderately inhibited by salt. Nonetheless, its activity over time is notoriously stabilized by salt. Furthermore, glycine betaine has a protective effect against KCl inhibition and enhances the thermal stability of both enzymes. The resurrection of the last common ancestor of ADP-PFK/GK from Methanosarcinales shows that the ancestral enzyme displays an extremely high salt tolerance and thermal stability. Structure determination of the ancestral protein reveals unique traits such as an increase in the Lys and Glu content at the protein surface and yet no reduction to the volume of the hydrophobic core. Our results suggest that the halophilic character is an ancient trait in the evolution of this protein family and that proteins from Methanosarcinales have adapted to highly saline environments by a non-canonical strategy, different from that currently proposed for Halobacteria. These results open up new avenues for the search and development of novel salt tolerant biocatalysts.
-
Image_5_ADP-Dependent Kinases From the Archaeal Order Methanosarcinales Adapt to Salt by a Non-canonical Evolutionarily Conserved Strategy.PDF
2018Co-Authors: Felipe Gonzalez-ordenes, Pablo Cea, Nicolás Fuentes-ugarte, Sebastián M. Muñoz, Ricardo A. Zamora, Diego Leonardo, Richard Charles Garratt, Victor Castro-fernandezAbstract:Halophilic organisms inhabit hypersaline environments where the extreme ionic conditions and osmotic pressure have driven the evolution of molecular adaptation mechanisms. Understanding such mechanisms is limited by the common difficulties encountered in cultivating such organisms. Within the Euryarchaeota, for example, only the Halobacteria and the order Methanosarcinales include readily cultivable halophilic species. Furthermore, only the former have been extensively studied in terms of their component proteins. Here, in order to redress this imbalance, we investigate the halophilic adaptation of glycolytic enzymes from the ADP-dependent phosphofructokinase/glucokinase family (ADP-PFK/GK) derived from organisms of the order Methanosarcinales. Structural analysis of proteins from non-halophilic and halophilic Methanosarcinales shows an almost identical composition and distribution of amino acids on both the surface and within the core. However, these differ from those observed in Halobacteria or Eukarya. Proteins from Methanosarcinales display a remarkable increase in surface lysine content and have no reduction to the hydrophobic core, contrary to the features ubiquitously observed in Halobacteria and which are thought to be the main features responsible for their halophilic properties. Biochemical characterization of recombinant ADP-PFK/GK from M. evestigatum (halophilic) and M. mazei (non-halophilic) shows the activity of both these extant enzymes to be only moderately inhibited by salt. Nonetheless, its activity over time is notoriously stabilized by salt. Furthermore, glycine betaine has a protective effect against KCl inhibition and enhances the thermal stability of both enzymes. The resurrection of the last common ancestor of ADP-PFK/GK from Methanosarcinales shows that the ancestral enzyme displays an extremely high salt tolerance and thermal stability. Structure determination of the ancestral protein reveals unique traits such as an increase in the Lys and Glu content at the protein surface and yet no reduction to the volume of the hydrophobic core. Our results suggest that the halophilic character is an ancient trait in the evolution of this protein family and that proteins from Methanosarcinales have adapted to highly saline environments by a non-canonical strategy, different from that currently proposed for Halobacteria. These results open up new avenues for the search and development of novel salt tolerant biocatalysts.
-
Table_2_ADP-Dependent Kinases From the Archaeal Order Methanosarcinales Adapt to Salt by a Non-canonical Evolutionarily Conserved Strategy.PDF
2018Co-Authors: Felipe Gonzalez-ordenes, Pablo Cea, Nicolás Fuentes-ugarte, Sebastián M. Muñoz, Ricardo A. Zamora, Diego Leonardo, Richard Charles Garratt, Victor Castro-fernandezAbstract:Halophilic organisms inhabit hypersaline environments where the extreme ionic conditions and osmotic pressure have driven the evolution of molecular adaptation mechanisms. Understanding such mechanisms is limited by the common difficulties encountered in cultivating such organisms. Within the Euryarchaeota, for example, only the Halobacteria and the order Methanosarcinales include readily cultivable halophilic species. Furthermore, only the former have been extensively studied in terms of their component proteins. Here, in order to redress this imbalance, we investigate the halophilic adaptation of glycolytic enzymes from the ADP-dependent phosphofructokinase/glucokinase family (ADP-PFK/GK) derived from organisms of the order Methanosarcinales. Structural analysis of proteins from non-halophilic and halophilic Methanosarcinales shows an almost identical composition and distribution of amino acids on both the surface and within the core. However, these differ from those observed in Halobacteria or Eukarya. Proteins from Methanosarcinales display a remarkable increase in surface lysine content and have no reduction to the hydrophobic core, contrary to the features ubiquitously observed in Halobacteria and which are thought to be the main features responsible for their halophilic properties. Biochemical characterization of recombinant ADP-PFK/GK from M. evestigatum (halophilic) and M. mazei (non-halophilic) shows the activity of both these extant enzymes to be only moderately inhibited by salt. Nonetheless, its activity over time is notoriously stabilized by salt. Furthermore, glycine betaine has a protective effect against KCl inhibition and enhances the thermal stability of both enzymes. The resurrection of the last common ancestor of ADP-PFK/GK from Methanosarcinales shows that the ancestral enzyme displays an extremely high salt tolerance and thermal stability. Structure determination of the ancestral protein reveals unique traits such as an increase in the Lys and Glu content at the protein surface and yet no reduction to the volume of the hydrophobic core. Our results suggest that the halophilic character is an ancient trait in the evolution of this protein family and that proteins from Methanosarcinales have adapted to highly saline environments by a non-canonical strategy, different from that currently proposed for Halobacteria. These results open up new avenues for the search and development of novel salt tolerant biocatalysts.
Wijaya Altekar - One of the best experts on this subject based on the ideXlab platform.
-
adaptive response of haloferax mediterranei to low concentrations of nacl 20 in the growth medium
Archives of Microbiology, 1997Co-Authors: Sandra E Dsouza, Wijaya Altekar, S F DsouzaAbstract:Halobacteria require 20-25% NaCl for optimal growth and lyse when the salt concentration falls below 10%. The response of Haloferax mediterranei cells to low concentrations of NaCl (< 20%) in the medium was studied. The cells adapted to and grew in concentrations of NaCl as low as 10% and survived in concentrations lower than 5%. The cells synthesised a red pigment, bacterioruberin, in response to stress caused by a low concentration of NaCl (< 20%).
-
characterization of 1 phosphofructokinase from halophilic archaebacterium haloarcula vallismortis
Biochimica et Biophysica Acta, 1994Co-Authors: Vidhya Rangaswamy, Wijaya AltekarAbstract:Abstract 1-Phosphofructokinase (EC 2.7.1.56) (1PFK) was purified and characterized for the first time from an archaebacterial halophile Haloarcula vallismortis. The purification procedure involving (NH4)2SO4 fractionation, (NH4)2SO4-mediated chromatography on Sepharose 4B, CM-cellulose chromatography, hydrophobic on phenyl Sepharose and adsorption chromatography on hydroxylapatite yielded a preparation with a specific activity of 128 and 100-fold purification. From gel filtration and sucrose density gradient ultracentrifugation, the apparent molecular mass of Halobacterial 1PFK was found as 76 ± 5 kDa. The Halobacterial 1PFK appears to be monomeric and the possibility of an unstable phosphoenzyme intermediate during its catalysis could not be ruled out. As in the case of many Halobacterial enzymes, the 1PFK was found to be halophilic and thermostable. Other catalytic features of Halobacterial 1PFK were similar to its counterparts from eubacterial sources.
-
characterisation and purification of ribulose bisphosphate carboxylase from heterotrophically grown halophilic archaebacterium haloferax mediterranei
FEBS Journal, 1994Co-Authors: Rema Rajagopalan, Wijaya AltekarAbstract:The CO2-fixing enzyme of Calvin cycle ribulose-1,5-bisphosphate-carboxylase/oxygenase has been isolated from a halophilic bacterium, Haloferax mediterranei grown heterotrophically. A homogeneous preparation was obtained from sonicated extract of the cells by three steps, resulting in a specific activity of 52 nmol · min−1· mg protein−1. The physicochemical and catalytic properties of the enzyme were studied. The Halobacterial ribulose-bisphosphate carboxylase is an oligomer of 54-kDa and 14-kDa subunits as detected by SDS/PAGE. By sucrose-density-gradient centrifugation, the molecular mass of the enzyme was estimated as approximately 500 kDa indicating a hexadecameric nature. No evidence for an additional form of the enzyme devoid of small subunits was obtained. The enzyme required Mg2+ for activity, KCl for activity and stability, and an optimal pH of 7.8. In contrast to many halophilic proteins, ribulose-bisphosphate carboxylase from H. mediterranei is not an acidic protein. From the comparison of amino acid composition of Halobacterial enzyme with its counterparts from a few eukaryotic and eubacterial sources, the SΔQ values showed that these proteins share some compositional similarities.
