NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning,sequence determination and overexpression in Escherichia coli

A gene encoding NADP-dependent Ds-threo-isocitrate dehydrogenase was isolated from Haloferax volcanii genomic DNA by using a combination of polymerase chain reaction and screening of a lambda EMBL3 library. Analysis of the nucleotide sequence revealed an open reading frame of 1260 bp encoding a protein of 419 amino acids with 45837 Da molecular mass. This sequence is highly similar to previously sequenced isocitrate dehydrogenases. In the alignment of the amino acid sequences with those from several archaeal and mesophilic NADP-dependent isocitrate dehydrogenases, the residues involved in dinucleotide binding and isocitrate binding are well conserved. We have developed methods for the expression in Escherichia coli and purification of the enzyme from H. volcanii. This expression was carried out in E. coli as inclusion bodies using the cytoplasmic expression vector pET3a. The enzyme was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing EDTA, MgCl(2) and 3 M NaCl. Maximal activity was obtained after several hours incubation at room temperature.     

Isocitrate dehydrogenases from Haloferax volcanii and Sulfolobus solfataricus: enzyme purification, characterisation and N-terminal sequence

Abstract A Pyrococcus woesei Eco RI DNA fragment (3400 bp) harbouring the gene fus for elongation factor 2 (EF-2) was cloned and almost completely sequenced. Unlike Methanococcus vannielii (which displays the 'str operon'-like fus and tuf gene context, 5'- rps 12- rps 7- fus-tuf -3⊃ and similar to Sulfolobus acidocaldarius and Desulfurococcus mobilis , the Pyrococcus fus gene (732 codons) is unlinked to the rps and tuf genes, and is immediately followed (57 bp intergenic spacing) by an ORF of 106 codons. Both ORFs are preceded by potential archaeal promoters located 52 bp (for fus ) and 37 bp (for ORF106) upstream of the putative start codons. The Pyrococcus EF-2(G) equivalent factor is somewhat closer to the eukaryal than to the bacterial homolog, and also shares with the former the C-terminal sequence required for ADP ribosylation of EF-2 by Diphtheria toxin.     

Improvement of two-dimensional gel electrophoresis proteome maps of the haloarchaeon Haloferax volcanii

Proteins of haloarchaea are remarkably unstable in low-ionic-strength solvents and tend to aggregate under standard two-dimensional (2-D) gel electrophoresis conditions, causing strong horizontal streaking. We have developed a new approach to generate 2-D maps of halophilic proteins which included washing cells with 1.5 M Tris-HCl buffer. In addition, proteins were precipitated with acetone, solubilized with urea and thiourea in the presence of the sulfobetaine detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), reduced with tributylphosphine (TBP), and separated with microrange strips of immobilized pH gradients (pH 3.9-5.1). This combination enabled the construction of highly reproducible 2-D maps of Haloferax volcanii proteins.     

Membrane binding of ribosomes occurs at SecYE-based sites in the Archaea Haloferax volcanii

Whereas ribosomes bind to membranes at eukaryal Sec61alphabetagamma and bacterial SecYEG sites, ribosomal membrane binding has yet to be studied in Archaea. Accordingly, functional ribosomes and inverted membrane vesicles were prepared from the halophilic archaea Haloferax volcanii. The ability of the ribosomes to bind to the membranes was determined using a flotation approach. Proteolytic pretreatment of the vesicles, as well as quantitative analyses, revealed the existence of a proteinaceous ribosome receptor, with the affinity of binding being comparable to that found in Eukarya and Bacteria. Inverted membrane vesicles prepared from cells expressing chimeras of SecE or SecY fused to a cytoplasmically oriented cellulose-binding domain displayed reduced ribosome binding due to steric hindrance. Pretreatment with cellulose drastically reduced ribosome binding to chimera-containing but not wild-type vesicles. Thus, as in Eukarya and Bacteria, ribosome binding in Archaea occurs at Sec-based sites. However, unlike the situation in the other domains of Life, ribosome binding in haloarchaea requires molar concentrations of salt. Structural information on ribosome-Sec complexes may provide insight into this high salt-dependent binding.     

Properties of the chaperonin complex from the halophilic archaeon Haloferax volcanii

The halophilic archaeon Haloferax volcanii has three genes encoding type II chaperonins, named cct1, cct2 and cct3. We show here that the three CCT proteins are all expressed but not to the same level. All three proteins are further induced on heat shock. The CCT proteins were purified by ammonium sulphate precipitation, sucrose gradient centrifugation and hydrophobic interaction chromatography. This procedure yields a high molecular mass complex (or complexes). The complex has ATPase activity, which is magnesium dependent, low salt-sensitive and stable to at least 75 degrees C. Activity requires high levels of potassium ions and was reduced in the presence of an increasing concentration of sodium ions.     

Molecular and Comparative Analysis of the Hyperthermostable Pyrococcus Furiosus Glutamate Dehydrogenase and its Gene

The gene for glutamate dehydrogenase (GDH) from Pyrococcus furiosus has been cloned, sequenced and expressed in Escherichia coli. Significant GDH activity could be detected in this host, allowing the further structure-function analysis of this hyperthermostable hexameric enzyme. The deduced primary sequence of the P. furiosus GDH was homologous to various bacterial, archaeal and eukaryal GDHs. Detailed comparative analysis of the primary sequences of these GDHs suggest that a decrease in Gly residues can be a general stabilizing feature of proteins functional under extreme conditions such as high temperatures or high salt concentrations whereas an increase in He residues and a decrease in Cys residues is typical for hyperthermostable enzymes.     

Insights into the molecular basis of thermal stability from the structure determination of Pyrococcus furiosus gluatamate dehydrogenase

Abstract: The structure determination of the glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus has been completed at 2.2 Å resolution. The structure has been compared with the glutamate dehydrogenases from the mesophiles Clostridium symbiosum, Escherichia coli and Neurospora crassa . This comparison has revealed that the hyperthermophilic enzyme contains a striking series of networks of ion-pairs which are formed by regions of the protein which contain a high density of charged residues. Such regions are not found in the mesophilic enzymes and the number and extent of ion-pair formation is much more limited. The ion-pair networks are clustered at both inter domain and inter subunit interfaces and may well represent a major stabilising feature associated with the adaptation of enzymes to extreme temperatures.     

Temperature-dependent acetoin production by Pyrococcus furiosus is catalyzed by a biosynthetic acetolactate synthase and its deletion improves ethanol production

The hyperthermophilic archaeon, Pyrococcus furiosus, grows optimally near 100 °C by fermenting sugars to acetate, carbon dioxide and molecular hydrogen as the major end products. The organism has recently been exploited to produce biofuels using a temperature-dependent metabolic switch using genes from microorganisms that grow near 70 °C. However, little is known about its metabolism at the lower temperatures. We show here that P. furiosus produces acetoin (3-hydroxybutanone) as a major product at temperatures below 80 °C. A novel type of acetolactate synthase (ALS), which is involved in branched-chain amino acid biosynthesis, is responsible and deletion of the als gene abolishes acetoin production. Accordingly, deletion of als in a strain of P. furiosus containing a novel pathway for ethanol production significantly improved the yield of ethanol. These results also demonstrate that P. furiosus is a potential platform for the biological production of acetoin at temperatures in the 70–80 °C range.     

A highly thermostable ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus

A ferritin from the obligate anaerobe and hyperthermophilic archaeon Pyrococcus furiosus (optimal growth at 100°C) has been cloned and overproduced in Escherichia coli to one-fourth of total cell-free extract protein, and has been purified in one step to homogeneity. The ferritin (PfFtn) is structurally similar to known bacterial and eukaryal ferritins; it is a 24-mer of 20 kDa subunits, which add up to a total Mr 480 kDa. The protein belongs to the non-heme type of ferritins. The 24-mer contains approximately 17 Fe (as isolated), 2,700 Fe (fully loaded), or <1 Fe (apoprotein). Fe-loaded protein exhibits an EPR spectrum characteristic for superparamagnetic core formation. At 25°C V_max=25 mole core Fe³⁺ formed per min per mg protein when measured at 315 nm, and the K_0.5=5 mM Fe(II). At 0.3 mM Fe(II) activity increases 100-fold from 25 to 85°C. The wild-type ferritin is detected in P. furiosus grown on starch. PfFtn is extremely thermostable; its activity has a half-life of 48 h at 100°C and 85 min at 120°C. No apparent melting temperature was found up to 120°C. The extreme thermostability of PfFtn has potential value for biotechnological applications.     

Straight-chain fatty alcohols in the hyperthermophilic archaeon Pyrococcus furiosus

Two straight-chain fatty alcohols (n-hexadecanol and n-octadecanol) were found in the neutral lipid fraction extracted from Pyrococcus furiosus cells. They were identified by thin-layer and gas-liquid chromatography, mass and infrared spectra, and chemical modification. The fatty alcohols accounted for 54% of the neutral lipid of the cell. Received: March 8, 2000 / Accepted: May 8, 2000     

Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough

A hyperthermophilic, anaerobic archaeon was isolated from hydrothermal fluid samples obtained at the Okinawa Trough vents in the NE Pacific Ocean, at a depth of 1395 m. The strain is obligately heterotrophic, and utilizes complex proteinaceous media (peptone, tryptone, or yeast extract), or a 21-amino-acid mixture supplemented with vitamins, as growth substrates. Sulfur greatly enhances growth. The cells are irregular cocci with a tuft of flagella, growing optimally at 98°C (maximum growth temperature 102°C), but capable of prolonged survival at 105°C. Optimum growth was at pH 7 (range 5–8) and NaCl concentration 2.4% (range 1%–5%). Tryptophan was required for growth, in contrast to the closely related strains Pyrococcus furiosus and P. abyssi. Thin sections of the cell, viewed by transmission electron microscopy, revealed a periplasmic space similar in appearance to the envelope of P. furiosus. The predominant cell membrane component was tetraether lipid, with minor amounts of diether lipids. Treatment of the cells by mild osmotic shock released an extract that contained a Zn²⁺-dependent alkaline phosphatase. Phylogenetic analysis of the sequences encoding 16S rRNA and glutamate dehydrogenase places the isolate with certainty within the genus Pyrococcus although there is relatively low DNA–DNA hybridization (<63%) with described species of this genus. Based on the reported results, we propose a new species, to be named Pyrococcus horikoshii sp. nov. Received: December 10, 1997 / Accepted: February 4, 1998     

Comprehensive Search for DNA Polymerase in the Hyperthermophilic Archaeon,Pyrococcus furiosus

DNA polymerase activities were scanned in a Pyrococcus furiosus cell extract to identify all of the DNA polymerases in this organism. Three main fractions containing DNA polymerizing activity were subjected to Western blot analyses, which revealed that the main activities in each fraction were derived from three previously identified DNA polymerases. PCNA (proliferating cell nuclear antigen), the sliding clamp of DNA polymerases, did not bind tightly to any of the three DNA polymerases. A primer usage preference was also shown for each purified DNA polymerase. Considering their biochemical properties, the roles of the three DNA polymerases during DNA replication in the cells are discussed.     

Characterization of Inverted Membrane Vesicles from the Halophilic Archaeon Haloferax volcanii

Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood. One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells. Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles. Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membrane-related phenomena in archaea. Received: 27 March 2001/Revised: 13 June 2001     

Crystal structure of an archaeal DNA sliding clamp: proliferating cell nuclear antigen from Pyrococcus furiosus

The proliferating cell nuclear antigen (PCNA) is now recognized as one of the key proteins in DNA metabolic events because of its direct interactions with many proteins involved in important cellular processes. We have determined the crystal structure of PCNA from a hyperthermophilic archaeon, Pyrococcus furiosus (pfuPCNA), at 2.1 A resolution. pfuPCNA forms a toroidal, ring-shaped structure consisting of homotrimeric molecules, which is also observed in the PCNA crystals from human and yeast. The overall structure of pfuPCNA is highly conserved with other PCNA proteins, as well as with the bacterial ss clamp and the bacteriophage gp45. This result shows that the three-dimensional structure of the sliding clamp is conserved in the three domains of life. pfuPCNA has two remarkable features compared with the human and yeast PCNA molecules: it has more ion pairs and fewer intermolecular main chain hydrogen bonds. The former may contribute to the thermal stability of pfuPCNA, and the latter may be the cause of the stimulatory effect of pfuPCNA on the DNA synthesizing activity of P. furiosus DNA polymerases in the absence of the clamp loader replication factor C in vitro.     

Haloferax volcanii AglB and AglD are involved in N-glycosylation of the S-layer glycoprotein and proper assembly of the surface layer

In this study, the effects of deleting two genes previously implicated in Haloferax volcanii N-glycosylation on the assembly and attachment of a novel Asn-linked pentasaccharide decorating the H. volcanii S-layer glycoprotein were considered. Mass spectrometry revealed the pentasaccharide to comprise two hexoses, two hexuronic acids and an additional 190 Da saccharide. The absence of AglD prevented addition of the final hexose to the pentasaccharide, while cells lacking AglB were unable to N-glycosylate the S-layer glycoprotein. In AglD-lacking cells, the S-layer glycoprotein-based surface layer presented both an architecture and protease susceptibility different from the background strain. By contrast, the absence of AglB resulted in enhanced release of the S-layer glycoprotein. H. volcanii cells lacking these N-glycosylation genes, moreover, grew significantly less well at elevated salt levels than did cells of the background strain. Thus, these results offer experimental evidence showing that N-glycosylation endows H. volcanii with an ability to maintain an intact and stable cell envelope in hypersaline surroundings, ensuring survival in this extreme environment.