Crystal structure of an archaeal peroxiredoxin from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1

Peroxiredoxins (Prxs) are thiol-dependent peroxidases that catalyze the detoxification of various peroxide substrates such as H2O2, peroxinitrite, and hydroperoxides, and control some signal transduction in eukaryotic cells. Prxs are found in all cellular organisms and represent an enormous superfamily. Recent genome sequencing projects and biochemical studies have identified a novel subfamily, the archaeal Prxs. Their primary sequences are similar to those of the 1-Cys Prxs, which use only one cysteine residue in catalysis, while their catalytic properties resemble those of the typical 2-Cys Prxs, which utilize two cysteine residues from adjacent monomers within a dimer in catalysis. We present here the X-ray crystal structure of an archaeal Prx from the aerobic hyperthermophilic crenarchaeon, Aeropyrum pernix K1, determined at 2.3 A resolution (Rwork of 17.8% and Rfree of 23.0%). The overall subunit arrangement of the A.pernix archaeal Prx is a toroid-shaped pentamer of homodimers, or an (alpha2)5 decamer, as observed in the previously reported crystal structures of decameric Prxs. The basic folding topology and the peroxidatic active site structure are essentially the same as those of the 1-Cys Prx, hORF6, except that the C-terminal extension of the A.pernix archaeal Prx forms a unique helix with its flanking loops. The thiol group of the peroxidatic cysteine C50 is overoxidized to sulfonic acid. Notably, the resolving cysteine C213 forms the intra-monomer disulfide bond with the third cysteine, C207, which should be a unique structural characteristic in the many archaeal Prxs that retain two conserved cysteine residues in the C-terminal region. The conformational flexibility near the intra-monomer disulfide linkage might be necessary for the dramatic structural rearrangements that occur in the catalytic cycle.     

Characterization of native glutamate dehydrogenase from an aerobic hyperthermophilic archaeon Aeropyrum pernix K1

Glutamate dehydrogenase (GDH) was purified and characterized from an aerobic hyperthermophilic archaeon Aeropyrum pernix (A. pernix) K1. The enzyme has a hexameric structure with a native molecular mass of about 285 +/- 15 kDa. It was specific for NADP and thermostable (74% activity was remained after 5 h incubation at 100 degrees C). The activity of the enzyme increased in the presence of polar water-miscible organic solvents such as acetonitrile, methanol, and ethanol. The N-terminal sequence of GDH is Met-Gln-Pro-Thr-Asp-Pro-Leu-Glu-Glu-Ala. This sequence, except for the methionine, corresponds to amino acids 7-15 of the open reading frame (ORF) encoding the predicted GDH (ORF APE 1386). In the ORF nucleotide sequence, the codon TTG appears at the position of the methionine, suggesting that the leucine codon might be recognized as an initiation codon and translated to methionine in A. pernix GDH.     

Isoprenoid quinones in an aerobic hyperthermophilic archaeon, Aeropyrum pernix

Aeropyrum pernix is the first strictly aerobic hyperthermophile known to grow heterotrophically at neutral pH and at temperatures up to 100°C. Using a simple and sensitive frit-fast atom bombardment liquid chromatography/mass spectrometry quinone analysis method, we analyzed the quinones in A. pernix. This organism contained demethylmenaquinone analogs (DMK-6(H_n)) and methionaquinone analogs (MTK-6(H_n)) when it was grown under vigorous shaking in the presence of air. The quinones were partially or fully saturated with six isoprenyl units. Although DMK and MTK are the quinones found in eubacteria, this is the first report to demonstrate the simultaneous occurrence of DMK and MTK in archaea. The effect of Na₂S₂O₃ on the quinone composition was studied at concentrations of 0, 0.1 and 0.5% under aerobic growth conditions with shaking. The total quinone content was highest (83.4 μg g⁻¹ dry cell weight) at 0.1% Na₂S₂O₃. In the absence of Na₂S₂O₃, only DMK-6 analogs were detected. While DMK analogs such as DMK-6(H₁₂), DMK-6(H₁₀) and DMK-6(H₈) were the major quinones at 0.1% Na₂S₂O₃, MTK analogs such as MTK-6(H₁₂) and MTK-6(H₁₀) were also detected. When the Na₂S₂O₃ concentration was increased to 0.5%, both DMK-6(H₈) and MTK-6(H₁₀) disappeared, while MTK-6(H₁₂) increased to approximately 20% of the total quinone content. When A. pernix was grown under oxygen limitation in a tightly closed bottle without gas phase, MK-6(H₁₀) appeared.     

Kinetic study of sn-glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1.

A gene having high sequence homology (45-49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94-96 degrees C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H-dependent dihydroxyacetone phosphate reduction and NAD(+)-dependent glycerol-1-phosphate (Gro1P) oxidation. NADP(+)-dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)(+) acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi-bi mechanism.     

A cambialistic SOD in a strictly aerobic hyperthermophilic archaeon, Aeropyrum pernix.

The superoxide dismutase (SOD) gene of Aeropyrum pernix, a strictly aerobic hyperthermophilic archaeon, was cloned and expressed in Escherichia coli, and its gene product was characterized. The molecular mass of the protein, based on the deduced amino acid sequence, was 24.6 kDa. The sequence showed overall similarity to the sequences of known Mn- and Fe-SODs. The metal binding residues conserved in Mn- and Fe-SODs were also found in A. pernix SOD. When the SOD gene was expressed in E. coli cells, the product formed a homodimer, and contained both Mn and Fe. Metal reconstitution experiments showed that A. pernix SOD is cambialistic, i.e. active with either Fe or Mn. The specific activities were 906 U/mg with Mn and 175 U/mg with Fe. No loss of activity of Mn-reconstituted SOD was observed at 105 degrees C even after 5 h incubation. Sodium azide, an inhibitor of SODs, did not inhibit the Mn-reconstituted SOD from A. pernix even at concentrations up to 400 mM. This SOD from an aerobic hyperthermophilic archaeon, Aeropyrum pernix, was extremely thermostable and active with either Mn or Fe. With Mn as a metal cofactor, it was more thermostable, and less sensitive to sodium azide and sodium fluoride than with Fe.     

Temperature dependence of kinetic parameters for hyperthermophilic glutamate dehydrogenase from Aeropyrum pernix K1

The temperature dependence of the steady-state kinetic parameters for a glutamate dehydrogenase from Aeropyrum pernix K1 was investigated. The enzyme showed a biphasic kinetic characteristic for L-glutamate and a monophasic one for NADP at 50-90 degrees C. At low concentrations of L-glutamate the Km decreased from 2.02 to 0.56 mM and the catalytic efficiency (Vmax/Km) markedly increased (4-150 micromol x mg(-1) x mM(-1)) along with the increase of temperature from 50 to 90 degrees C. At high concentrations of the substrate the Km was fairly high and approximately constant (around 225 mM), and the catalytic efficiency was low and its temperature-dependent change was small. The Km (0.039 mM) for NADP did not change with the increase of temperature. In the reductive amination, the Kms for 2-oxoglutarate (1.81 and 9.37 mM at low and high levels of ammonia, respectively) were independent on temperature, but the Kms for ammonia and NADPH rose from 86 to 185 mM and 0.050 to 0.175 mM, respectively.     

Provirus induction in hyperthermophilic archaea: characterization of Aeropyrum pernix spindle-shaped virus 1 and Aeropyrum pernix ovoid virus 1

By in silico analysis, we have identified two putative proviruses in the genome of the hyperthermophilic archaeon Aeropyrum pernix, and under special conditions of A. pernix growth, we were able to induce their replication. Both viruses were isolated and characterized. Negatively stained virions of one virus appeared as pleomorphic spindle-shaped particles, 180 to 210 nm by 40 to 55 nm, with tails of heterogeneous lengths in the range of 0 to 300 nm. This virus was named Aeropyrum pernix spindle-shaped virus 1 (APSV1). Negatively stained virions of the other virus appeared as slightly irregular oval particles with one pointed end, while in cryo-electron micrographs, the virions had a regular oval shape and uniform size (70 by 55 nm). The virus was named Aeropyrum pernix ovoid virus 1 (APOV1). Both viruses have circular, double-stranded DNA genomes of 38,049 bp for APSV1 and 13,769 bp for APOV1. Similarities to proteins of other archaeal viruses were limited to the integrase and Dna1-like protein. We propose to classify APOV1 into the family Guttaviridae.     

A novel phospholipase A2/esterase from hyperthermophilic archaeon Aeropyrum pernix K1

An open reading frame of the hyperthermophilic archaeon Aeropyrum pernix K1 APE2325, which composed of 474 bases, was cloned and expressed in Escherichia coli BL21 (DE3) Codon Plus-RIL. The recombinant protein was purified by Ni-chelation affinity chromatography. It showed a single band with a molecular mass of 18kDa in SDS-PAGE. The purified enzyme exhibited both phospholipase A(2) and esterase activities with the optimal catalytic temperature at 90 degrees C. The enzyme activity was Ca(2+)-independent. Kinetic analysis revealed its Km, k cat, and Vm for the p-nitrophenyl propionate substrate were 103microM, 39s(-1), and 249micromol/min/mg, respectively. The recombinant protein was thermostable and its half-life at 100 degrees C was about 1h.     

Gene recognition based on nucleotide distribution of ORFs in a hyper-thermophilic crenarchaeon, Aeropyrum pernix K1.

The 2694 ORFs originally annotated as potential genes in the genome of Aeropyrum pernix can be categorized into three clusters (A, B, C), according to their nucleotide composition at three codon positions. Coding potential was found to be responsible for the phenomenon of three clusters in a 9-dimensional space derived from the nucleotide composition of ORFs: ORFs assigned to cluster A are coding ones, while those assigned to clusters B and C are non-coding ORFs. A "codingness" index called the AZ score is defined based on a clustering method used to recognize protein-coding genes in the A. pernix genome. The criterion for a coding or non-coding ORF is based on the AZ score. ORFs with AZ > 0 or AZ < 0 are coding or non-coding, respectively. Consequently, 620 out of 632 ORFs with putative functions based on the original annotation are contained in cluster A, which have positive AZ scores. In addition, all 29 ORFs encoding putative or conserved proteins newly added in RefSeq annotation also have positive AZ scores. Accordingly, the number of re-recognized protein-coding genes in the A. pernix genome is 1610, which is significantly less than 2694 in the original annotation and also much less than 1841 in the RefSeq annotation curated by NCBI staff. Annotation information of re-recognized genes and their AZ scores are available at: http://tubic.tju.edu.cn/Aper/.     

Aeropyrum pernix K1, a strictly aerobic and hyperthermophilic archaeon,has two terminal oxidases,cytochrome ba3 and cytochrome aa3

Aeropyrum pernix K1 is a strictly aerobic and hyperthermophilic archaeon that thrives even at 100 degrees C. The archaeon is quite interesting with respect to the evolution of aerobic electron transport systems and the thermal stability of the respiratory components. An isolated membrane fraction was found to oxidize bovine cytochrome c.The activity was solubilized in the presence of detergents and separated into two fractions by successive chromatography. Two cytochrome oxidases, designated as CO-1 and CO-2, were further purified. CO-1 was a ba(3)-type cytochrome containing at least two subunits. Chemically digested fragments of CO-1 revealed a peptide with a sequence identical to a part of a putative cytochrome oxidase subunit I encoded by the gene ape1623. CO-2, an aa(3)-type cytochrome, was present in lower amounts than CO-1 and was immunologically identified as a product of aoxABC gene (DDBJ accession no. AB020482). Both cytochromes reacted with carbon monoxide. The apparent K(m) values of CO-1 and CO-2 for oxygen were 5.5 and 32 micro M, respectively, at 25 degrees C. The terminal oxidases CO-1 and CO-2 phylogenetically correspond to the SoxB and SoxM branches, respectively, of the heme-copper oxidase tree.     

Structural analysis of the substrate recognition mechanism in O-phosphoserine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1

L-Cysteine is synthesized from O-acetyl-L-serine (OAS) and sulfide by O-acetylserine sulfhydrylase (OASS; EC 2.5.1.47) in plants and bacteria. O-phosphoserine sulfhydrylase (OPSS; EC 2.5.1.65) is a novel enzyme from the hyperthermophilic aerobic archaeon Aeropyrum pernix K1 (2003). OPSS can use OAS or O-phospho-L-serine (OPS) to synthesize L-cysteine. To elucidate the mechanism of the substrate specificity of OPSS, we analyzed three-dimensional structures of the active site of the enzyme. The active-site lysine (K127) of OPSS forms an internal Schiff base with pyridoxal 5'-phosphate. Therefore, crystals of the complexes formed by the K127A mutant with the external Schiff base of pyridoxal 5'-phosphate with either OPS or OAS were prepared and examined by X-ray diffraction analysis. In contrast to that observed for OASS, no significant difference was seen in the overall structure between the free and complexed forms of OPSS. The side chains of T152, S153, and Q224 interacted with the carboxylate of the substrates, as a previous study has suggested. The side chain of R297 has been proposed to recognize the phosphate group of OPS. Surprisingly, however, the position of R297 was significantly unchanged in the complex of the OPSS K127A mutant with the external Schiff base, allowing enough space for an interaction with OPS. The positively charged environment around the entrance of the active site including S153 and R297 is important for accepting negatively charged substrates such as OPS.     

Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1: enzymatic characterization, identification of the encoding gene, and phylogenetic implications

NADP-dependent glutamate dehydrogenase (l-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) was purified to homogeneity for characterization. The enzyme retained its full activity on heating at 95°C for 30 min, and the maximum activity in l-glutamate deamination was obtained around 100°C. The enzyme showed a strict specificity for l-glutamate and NADP on oxidative deamination and for 2-oxoglutarate and NADPH on reductive amination. The K _m values for NADP, l-glutamate, NADPH, 2-oxoglutarate, and ammonia were 0.039, 3.3, 0.022, 1.7, and 83 mM, respectively. On the basis of the N-terminal amino acid sequence, the encoding gene was identified in the A. pernix K1 genome, cloned, and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1257 bp starting with a minor TTG codon and encoding a protein of 418 amino acids with a molecular weight of 46 170. Phylogenetic analysis revealed that the glutamate dehydrogenase from A. pernix K1 clustered with those from aerobic Sulfolobus solfataricus, Sulfolobus shibatae, and anaerobic Pyrobaculum islandicum in Crenarchaeota, and it separated from another cluster of the enzyme from Thermococcales in Euryarchaeota. The branching pattern of the enzymes from A. pernix K1, S. solfataricus, S. shibatae, and Pb. islandicum in the phylogenetic tree coincided with that of 16S rDNAs obtained from the same organisms. Received: April 24, 2000 / Accepted: August 10, 2000     

Properties of the alpha subunit of a Chaperonin from the hyperthermophilic Crenarchaeon Aeropyrum pernix K1

The gene encoding for a putative thermosome from the hyperthermophilic crenarchaeon Aeropyrum pernix K1 (ApcpnA) was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE0907) from A. pernix K1 showed some homology with other group II chaperonins from archaea. The recombinant ApcpnA protein has a molecular mass of 60 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and exhibited ATPase activity with an optimum temperature and pH of 90 degrees C and 5.0, respectively. The ATPase activity was found to be dependent on manganese and potassium ions, but not magnesium ion. The K(m) for ATP at pH 5.0 and 90 degrees C was 10.04 (+/- 1.31) microM, and k(cat) was determined to be 2.21 (+/- 0.11) min(-1) for the ApcpnA monomer. The recombinant ApcpnA prevents thermal aggregation of bovine rhodanese and enhances the thermal stability of alcohol dehydrogenase in vitro, indicating that the protein is suitable as a molecular chaperonin in the high-temperature environment.     

Stability of diether C(25,25) liposomes from the hyperthermophilic archaeon Aeropyrum pernix K1

Temperature and pH effects were studied for stability, structural organization, fluidity and permeability of vesicles from a polar lipid methanol fraction isolated from the Aeropyrum pernix. We determined the permeability of C_25,25 liposomes using fluorescence intensity of released calcein. At pH 7.0 and 9.0, and from 85 °C to 98 °C, only 10% of entrapped calcein was released. After 10 h at 90 °C, calcein release reached 27%, independent of pH. Fluorescence anisotropy measurements of hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene revealed gradual changes up to 60 °C. At higher temperatures, the anisotropy did not change significantly. Fluorescence alone did not provide detailed and direct structural information about these C_25,25 liposomes, so we used electron paramagnetic resonance spectroscopy (EPR) and differential scanning calorimetry (DSC). From EPR spectra, mean membrane fluidity determined according to maximal hyperfine splitting and empirical correlation times showed continuous increases with temperature. Computer simulation of EPR spectra showed heterogeneous membranes of these C_25,25 liposomes: at low temperatures, they showed three types of membrane regions characterized by different motional modes. Above 65 °C, the membrane becomes homogeneous with only one fluid-like region. DSC thermograms of C_25,25 liposomes reveal a very broad and endothermic transition in the temperature range from 0 °C to 40 °C.     

Complete Genome Sequence of an Aerobic Hyper-thermophilic Crenarchaeon, Aeropyrum pernix K1

The complete sequence of the genome of an aerobic hyper-thermophiliccrenarchaeon, Aeropyrum pernix K1, which optimally grows at95°C, has been determined by the whole genome shotgun methodwith some modifications. The entire length of the genome was1,669,695 bp. The authenticity of the entire sequence was supportedby restriction analysis of long PCR products, which were directlyamplified from the genomic DNA. As the potential protein-codingregions, a total of 2,694 open reading frames (ORFs) were assigned.By similarity search against public databases, 633 (23.5%) ofthe ORFs were related to genes with putative function and 523(19.4%) to the sequences registered but with unknown function.All the genes in the TCA cycle except for that of alpha-ketoglutaratedehydrogenase were included, and instead of the alpha-ketoglutaratedehydrogenase gene, the genes coding for the two subunits of2-oxoacid:ferredoxin oxidoreductase were identified. The remaining1,538 ORFs (57.1%) did not show any significant similarity tothe sequences in the databases. Sequence comparison among theassigned ORFs suggested that a considerable member of ORFs weregenerated by sequence duplication. The RNA genes identifiedwere a single 16S–23S rRNA operon, two 5S rRNA genes and47 tRNA genes including 14 genes with intron structures. Allthe assigned ORFs and RNA coding regions occupied 89.12% ofthe whole genome. The data presented in this paper are availableon the internet homepage (http://www.mild.nite.go.jp).     

The structure of an alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix

The structure of the recombinant medium chain alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Aeropyrum pernix has been solved by the multiple anomalous dispersion technique using the signal from the naturally occurring zinc ions. The enzyme is a tetramer with 222 point group symmetry. The ADH monomer is formed from a catalytic and a cofactor-binding domain, with the overall fold similar to previously solved ADH structures. The 1.62 A resolution A.pernix ADH structure is that of the holo form, with the cofactor NADH bound into the cleft between the two domains. The electron density found in the active site has been interpreted to be octanoic acid, which has been shown to be an inhibitor of the enzyme. This inhibitor is positioned with its carbonyl oxygen atom forming the fourth ligand of the catalytic zinc ion. The structural zinc ion of each monomer is present at only partial occupancy and in its absence a disulfide bond is formed. The enhanced thermal stability of the A.pernix ADH is thought to arise primarily from increased ionic and hydrophobic interactions on the subunit interfaces.     

Cooperativity of alpha- and beta-subunits of group II chaperonin from the hyperthermophilic archaeum Aeropyrum pernix K1

alpha- and beta-subunits (ApCpnA and ApCpnB) are group II chaperonins from the hyperthermophilic archaeum Aeropyrum pernix K1, specialized in preventing the aggregation and inactivation of substrate proteins under conditions of transient heat stress. In the present study, the cooperativity of alpha- and beta-subunits from the A. pernix K1 was investigated. The ApCpnA and ApCpnB chaperonin genes were overexpressed in E. coli Rosetta and Codonplus (DE3), respectively. Each of the recombinant alpha- and beta- subunits was purified to 92% and 94% by using anionexchange chromatography. The cooperative activity between purified alpha- and beta-subunits was examined using citrate synthase (CS), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) as substrate proteins. The addition of both alpha- and beta-subunits could effectively protect CS and ADH from thermal aggregation and inactivation at 43 degreesC and 50 degreesC, respectively, and MDH from thermal inactivation at 80 degreesC and 85 degreesC. Moreover, in the presence of ATP, the protective effects of alpha- and beta-subunits on CS from thermal aggregation and inactivation, and ADH from thermal aggregation, were more enhanced, whereas cooperation between chaperonins and ATP in protection activity on ADH and MDH (at 85 degreesC) from thermal inactivation was not observed. Specifically, the presence of both alpha- and beta- subunits could effectively protect MDH from thermal inactivation at 80 degreesC in an ATP-dependent manner.     

Inhibitory effect of Maillard reaction products on growth of the aerobic marine hyperthermophilic archaeon Aeropyrum pernix

It was found that the growth of Aeropyrum pernix was severely inhibited in a medium containing reducing sugars and tryptone due to the formation of Maillard reaction products. The rate of the Maillard browning reaction was markedly enhanced under aerobic conditions, and the addition of Maillard reaction products to the culture medium caused fatal growth inhibition.