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).     

Expression, purification and crystal structure of a truncated acylpeptide hydrolase from Aeropyrum pernix K1

Acylpeptide hydrolase （APH） catalyzes the N-terminal hydrolysis of N^α-acylpeptides to release N^α-acylated amino acids. The crystal structure of recombinant APH from the thermophilic archaeon Aeropyrum pernix K1 （apAPH） was reported recently to be at a resolution of 2.1 A using X-ray diffraction. A truncated mutant of apAPH that lacks the first short α-helix at the N-terminal, apAPH-△（1-21）, was cloned, expressed, characterized and crystallized. Data from biochemical experiments indicate that the optimum temperature of apAPH is decreased by 15℃ with the deletion of the N-terminal α-helix. However, the enzyme activity at the optimal temperature does not change. It suggests that this N-terminal α-helix is essential for thermostability. Here, the crystal structure of apAPH-△（1-21） has been determined by molecular replacement to 2.5 A. A comparison between the two structures suggests a difference in thermostability, and it can be concluded that by adding or deleting a linking structure （located over different domains）, the stability or even the activity of an enzyme can be modified.     

超嗜热需氧古生菌K1嗜热磷脂酶A2的序列和结构预测

目的:从超嗜热需氧古生菌(Aeropyrumpernix)K1中抽提染色体基因组,经PCR扩增获得磷脂酶A2基因,在大肠杆菌中诱导表达嗜热磷脂酶A2(APEPLA2),分析其氨基酸组成,预测其结构,从而探讨可能导致嗜热酶热稳定性的原因。方法:利用DNASIS二级结构预测软件和由NCBI提供的多种蛋白质进行同源性分析和结构预测。结果:同源序列分析表明APEPLA2与其他磷脂酶A2的同源性较低。结论:推测APEPLA2属于钙离子不依赖性(iPLA2),形成了以β折叠为中心、两边有多个长度不等的α螺旋环绕的α/β拓扑结构。     

Aeropyrum pernix K1磷脂酶A2的克隆、表达及其性质

从超嗜热需氧古细菌AeropyrumpernixK1中抽提出染色体基因组,经PCR扩增得到磷脂酶A2基因,用带有His-tag标记的pET15b作为表达载体,在大肠杆菌BLP中成功地诱导表达。表达产物经过Ni-螯合柱一步得到纯化。SDS-PAGE检测只有一条带,其准确分子量为17,871kD。对纯化后的磷脂酶A2测定其酶活性和生物活性,得出其最适反应温度为90℃,最适pH范围为7·8~8·2。至此首次成功地在大肠杆菌中表达了古细菌嗜热磷脂酶A2,这将为以后对该酶的结构和功能以及耐热机制研究打下很好的基础,同时有利于古细菌研究领域的扩展。     

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.     

Characterization of a Whole Set of tRNA Molecules in an Aerobic Hyper-thermophilic Crenarchaeon, Aeropyrum pernix K1

The tRNA molecule has an important role in translation, thefunction of which is to carry amino acids to the ribosomes.It is known that tRNA is transcribed from tRNA genes, some ofwhich, in Eukarya and Archaea, contain introns. A computationalanalysis of the complete genome of Aeropyrum pernix K1 predictedthe presence of 14 intron-containing tRNA genes. To elucidatewhether these introns are actually processed in living cellsand what mechanism detects the intron regions, cDNAs for prematureand mature forms of the tRNA molecules transcribed from theintron-containing tRNA genes in the model aerobic acidothermophiliccrenarchaeon, A. pernix K1 were identified and analyzed. A comparisonbetween the nucleotide sequences of these two types of cDNAsindicated that the intron regions of the tRNA molecules wereindeed processed in A. pernix K1 living cells. Some cDNA clonesshowed that the actual splicing positions were different fromthose predicted by computational analysis. However, the bulge–helix–bulgestructure, which has been previously identified in exon–intronboundaries of archaeal tRNA genes, was evident in all boundaryregions confirmed in this work. These results indicate thatthe generally described mechanism for tRNA processing in Archaeais utilized for processing the intron region of the tRNA moleculesin A. pernix K1.     

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     

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/.     

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.     

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.     

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.     

Proteome analysis of an aerobic hyperthermophilic crenarchaeon, Aeropyrum pernix K1

We analyzed the proteome of a crenararchaeon, Aeropyrum pernix K1, by using the following four methods: (i) two-dimensional PAGE followed by MALDI-TOF MS, (ii) one-dimensional SDS-PAGE in combination with two-dimensional LC-MS/MS, (iii) multidimensional LC-MS/MS, and (iv) two-dimensional PAGE followed by amino-terminal amino acid sequencing. These methods were found to be complementary to each other, and biases in the data obtained in one method could largely be compensated by the data obtained in the other methods. Consequently a total of 704 proteins were successfully identified, 134 of which were unique to A. pernix K1, and 19 were not described previously in the genomic annotation. We found that the original annotation of the genomic data of this archaeon was not adequate in particular with respect to proteins of 10-20 kDa in size, many of which were described as hypothetical. Furthermore the amino-terminal amino acid sequence analysis indicated that surprisingly the translation of 52% of their genes starts with TTG in contrast to ATG (28%) and GTG (20%). Thus, A. pernix K1 is the first example of an organism in which TTG is the most predominant translational initiation codon.     

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.     

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.     

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.     

Purification and characterization of an intracellular heat-stable proteinase (pernilase) from the marine hyperthermophilic archaeon Aeropyrum pernix K1

A novel intracellular serine proteinase from the marine aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) that we designated pernilase was purified by ammonium sulfate precipitation, anionic-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 50 kDa as determined by SDS-PAGE. The proteinase had a broad pH profile (pH 5–10) with an optimum pH of 9.0 for peptide hydrolysis. The optimum temperature for enzyme activity was 90°C. The enzyme was strongly inhibited by diisopropyl fluorophosphate (DFP) and phenylmethyl sulfonylfluoride (PMSF), suggesting that it corresponds to a serine proteinase. The enzyme was highly resistant to the reducing agents dithiothreitol and 2-mercaptoethanol but sensitive to the denaturing reagents guanidine-HCl and urea and also to the detergent sodium dodecyl sulfate (SDS). Pernilase showed high substrate specificity for Boc-Leu-Gly-Arg-MCA peptide. Thermostability of this enzyme showed half-lives of 85 min at 100°C and 12 min at 110°C. Received September 24, 1997 / Accepted May 20, 1998     

Catalytic properties and crystal structure of thermostable NAD(P)H-dependent carbonyl reductase from the hyperthermophilic archaeon Aeropyrum pernix K1

A gene encoding NAD(P)H-dependent carbonyl reductase (CR) from the hyperthermophilic archaeon Aeropyrum pernix K1 was overexpressed in Escherichia coli. Its product was effectively purified and characterized. The expressed enzyme was the most thermostable CR found to date; the activity remained at approximately 75% of its activity after incubation for 10 min up to 90 °C. In addition, A. pernix CR exhibited high stability at a wider range of pH values and longer periods of storage compared with CRs previously identified from other sources. A. pernix CR catalyzed the reduction of various carbonyl compounds including ethyl 4-chloro-3-oxobutanoate and 9,10-phenanthrenequinone, similar to the CR from thyroidectomized (Tx) chicken fatty liver. However, A. pernix CR exhibited significantly higher K_m values against several substrates than Tx chicken fatty liver CR. The three-dimensional structure of A. pernix CR was determined using the molecular replacement method at a resolution of 2.09 Å, in the presence of NADPH. The overall fold of A. pernix CR showed moderate similarity to that of Tx chicken fatty liver CR; however, A. pernix CR had no active-site lid unlike Tx chicken fatty liver CR. Consequently, the active-site cavity in the A. pernix CR was much more solvent-accessible than that in Tx chicken fatty liver CR. This structural feature may be responsible for the enzyme’s lower affinity for several substrates and NADPH. The factors contributing to the much higher thermostability of A. pernix CR were analyzed by comparing its structure with that of Tx chicken fatty liver CR. This comparison showed that extensive formation of the intrasubunit ion pair networks, and the presence of the strong intersubunit interaction, is likely responsible for A. pernix CR thermostability. Site-directed mutagenesis showed that Glu99 plays a major role in the intersubunit interaction. This is the first report regarding the characteristics and three-dimensional structure of hyperthermophilic archaeal CR.