Sequential aldol condensation catalyzed by hyperthermophilic 2-deoxy-d-ribose-5-phosphate aldolase

Genes encoding 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, were expressed individually in Escherichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E. coli enzyme, even at a low temperature (25 degrees C), although both enzymes showed much less 2-deoxy-d-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25 degrees C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 A. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs.     

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.     

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.     

Identification of the first archaeal oligopeptide-binding protein from the hyperthermophile Aeropyrum pernix

The archaeon Aeropyrum pernix grows optimally at 90°C and derives energy primarily from aerobic degradation of complex proteinaceous substrates. The ability of these nutrients to sustain growth is generally associated with the presence of oligopeptide transport systems, such as the well-known protein-dependent ATP-binding cassette (ABC) transporters. This study is concerned with the isolation and characterisation of the first archaeal oligopeptide-binding protein (OppA_Ap) from the extracellular medium of A. pernix. The protein shows a pI of 3.9 and a molecular mass of about 90 kDa under native conditions. By using a proteomic approach, the OppA_Ap-encoding gene was identified (APE1583) and about 55% of the protein amino-acid sequence was validated. The extracellular purified protein was able to efficiently bind oligopeptide substrates such as Xenopsin. The amount of a liganded peptide to OppA_Ap was about 70% at 90°C using a 1/100 (w/w) OppA_Ap/substrate ratio. Sequence comparisons showed a weak but significant similarity of OppA_Ap with bacterial oligopeptide binding proteins. Furthermore, APE1583 neighbouring genes encode for the cognate components of an ABC transport system, suggesting that these ORFs are organised in an operon-like structure, with OppA_Ap as the extracellular component for the uptake of oligopeptides.     

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.     

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.     

Crystal structure of archaeal chromatin protein Alba2-double-stranded DNA complex from Aeropyrum pernix K1

All thermophilic and hyperthermophilic archaea encode homologs of dimeric Alba (Sac10b) proteins that bind cooperatively at high density to DNA. Here, we report the 2.0 Å resolution crystal structure of an Alba2 (Ape10b2)-dsDNA complex from Aeropyrum pernix K1. A rectangular tube-like structure encompassing duplex DNA reveals the positively charged residues in the monomer-monomer interface of each dimer packing on either side of the bound dsDNA in successive minor grooves. The extended hairpin loop connecting strands β3 and β4 undergoes significant conformational changes upon DNA binding to accommodate the other Alba2 dimer during oligomerization. Mutational analysis of key interacting residues confirmed the specificity of Alba2-dsDNA interactions.     

Comparative analysis of pyruvate kinases from the hyperthermophilic archaea Archaeoglobus fulgidus,Aeropyrum pernix,and Pyrobaculum aerophilum and the hyperthermophilic bacterium Thermotoga maritima: unusual regulatory properties in hyperthermophilic archaea

Pyruvate kinases (PK, EC 2.7.1.40) from three hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the hyperthermophilic bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+. Temperature optima for activity were 85 degrees C (A. fulgidus) and above 98 degrees C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 degrees C (A. pernix) and showed melting temperatures for thermal unfolding at 93 degrees C (A. fulgidus) or above 98 degrees C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K+ ions, had a temperature optimum of 80 degrees C, was highly thermostable up to 90 degrees C, and had a melting temperature above 98 degrees C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the hyperthermophilic bacterium T. maritima.     

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.     

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.     

Crystal structure of translation initiation factor 5B from the crenarchaeon Aeropyrum pernix

Initiation factor 5B (IF5B) is a universally conserved translational GTPase that catalyzes ribosomal subunit joining. In eukaryotes, IF5B directly interacts via a groove in its domain IV with initiation factor 1A (IF1A), another universally conserved initiation factor, to accomplish efficient subunit joining. Here, we have determined the first structure of a crenarchaeal IF5B, which revealed that the archaea‐specific region of IF5B (helix α15) binds and occludes the groove of domain IV. Therefore, archaeal IF5B cannot access IF1A in the same manner as eukaryotic IF5B. This fact suggests that different relationships between IF5B and IF1A exist in archaea and eukaryotes. Proteins 2016; 84:712–717. © 2016 Wiley Periodicals, Inc.     

Crystal structure of an archaeal specific DNA-binding protein (Ape10b2) from Aeropyrum pernix K1

DNA binding proteins are essential in all organisms, and they play important roles in both compacting and regulating the genetic material. All thermophilic and hyperthermophilic archaea encode one or more copies of Alba or Sso10b, which is a small, abundant, basic protein that binds DNA. Here, we present the crystal structure of Ape10b2 from Aeropyrum pernix K1 at 1.70 A. Although the overall structure resembles the known Alba protein fold, a significant conformational change was observed in the loop regions. Specifically, the L5 loop is slightly longer, as compared to those of other known proteins, and the flexibility of this loop may facilitate the interaction with double stranded DNA. In addition, we showed that Ape10b2 binds to 16 and 39 bp duplex DNAs with high affinity. On the basis of our analyses, we have created a putative protein-DNA complex model.     

Crystal structure of the RNA 2'-phosphotransferase from Aeropyrum pernix K1

In the final step of tRNA splicing, the 2'-phosphotransferase catalyzes the transfer of the extra 2'-phosphate from the precursor-ligated tRNA to NAD. We have determined the crystal structure of the 2'-phosphotransferase protein from Aeropyrum pernix K1 at 2.8 Angstroms resolution. The structure of the 2'-phosphotransferase contains two globular domains (N and C-domains), which form a cleft in the center. The N-domain has the winged helix motif, a subfamily of the helix-turn-helix family, which is shared by many DNA-binding proteins. The C-domain of the 2'-phosphotransferase superimposes well on the NAD-binding fold of bacterial (diphtheria) toxins, which catalyze the transfer of ADP ribose from NAD to target proteins, indicating that the mode of NAD binding by the 2'-phosphotransferase could be similar to that of the bacterial toxins. The conserved basic residues are assembled at the periphery of the cleft and could participate in the enzyme contact with the sugar-phosphate backbones of tRNA. The modes by which the two functional domains recognize the two different substrates are clarified by the present crystal structure of the 2'-phosphotransferase.     

Structure and characterization of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PS(Ap)) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PS(Ap) would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PS(Ap) in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PS(Ap) is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PS(Ap) follows Michaelis-Menten kinetics at 60 °C, with a K(M) for erythrose 4-phosphate of 280 μM, a K(M) for phosphoenolpyruvate of 891 μM, and a k(cat) of 1.0 s(-1). None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PS(Ap). The structure of DAH7PS(Ap) is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.     

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.     

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.