The modified mevalonate pathway is believed to be the upstream biosynthetic route for isoprenoids in general archaea. The partially identified pathway has been proposed to explain a mystery ...surrounding the lack of phosphomevalonate kinase and diphosphomevalonate decarboxylase by the discovery of a conserved enzyme, isopentenyl phosphate kinase. Phosphomevalonate decarboxylase was considered to be the missing link that would fill the vacancy in the pathway between mevalonate 5-phosphate and isopentenyl phosphate. This enzyme was recently discovered from haloarchaea and certain Chroloflexi bacteria, but their enzymes are close homologs of diphosphomevalonate decarboxylase, which are absent in most archaea. In this study, we used comparative genomic analysis to find two enzymes from a hyperthermophilic archaeon, Aeropyrum pernix, that can replace phosphomevalonate decarboxylase. One enzyme, which has been annotated as putative aconitase, catalyzes the dehydration of mevalonate 5-phosphate to form a previously unknown intermediate, trans-anhydromevalonate 5-phosphate. Then, another enzyme belonging to the UbiD-decarboxylase family, which likely requires a UbiX-like partner, converts the intermediate into isopentenyl phosphate. Their activities were confirmed by in vitro assay with recombinant enzymes and were also detected in cell-free extract from A. pernix. These data distinguish the modified mevalonate pathway of A. pernix and likely, of the majority of archaea from all known mevalonate pathways, such as the eukaryote-type classical pathway, the haloarchaea-type modified pathway, and another modified pathway recently discovered from Thermoplasma acidophilum.
Thermostable nucleoside phosphorylases are attractive biocatalysts for the synthesis of modified nucleosides. Hence we report on the recombinant expression of three ‘high molecular mass’ purine ...nucleoside phosphorylases (PNPs) derived from the thermophilic bacteria Deinococcus geothermalis, Geobacillus thermoglucosidasius and from the hyperthermophilic archaeon Aeropyrum pernix (5′–methythioadenosine phosphorylase; ApMTAP). Thermostability studies, kinetic analysis and substrate specificities are reported. The PNPs were stable at their optimal temperatures (DgPNP, 55 °C; GtPNP, 70 °C; ApMTAP, activity rising to 99 °C). Substrate properties were investigated for natural purine nucleosides adenosine, inosine and their C2′‐deoxy counterparts (activity within 50–500 U·mg−1), analogues with 2′‐amino modified 2′‐deoxy‐adenosine and ‐inosine (within 0.1–3 U·mg−1) as well as 2′‐deoxy‐2′‐fluoroadenosine (9) and its C2′‐arabino diastereomer (10, within 0.01–0.03 U·mg−1). Our results reveal that the structure of the heterocyclic base (e.g. adenine or hypoxanthine) can play a critical role in the phosphorolysis reaction. The implications of this finding may be helpful for reaction mechanism studies or optimization of reaction conditions. Unexpectedly, the diastereomeric 2′‐deoxyfluoro adenine ribo‐ and arabino‐nucleosides displayed similar substrate properties. Moreover, cytidine and 2′‐deoxycytidine were found to be moderate substrates of the prepared PNPs, with substrate activities in a range similar to those determined for 2′‐deoxyfluoro adenine nucleosides 9 and 10. C2′‐modified nucleosides are accepted as substrates by all recombinant enzymes studied, making these enzymes promising biocatalysts for the synthesis of modified nucleosides. Indeed, the prepared PNPs performed well in preliminary transglycosylation reactions resulting in the synthesis of 2′‐deoxyfluoro adenine ribo‐ and arabino‐ nucleosides in moderate yield (24%).
Recombinant purine nucleoside phosphorylases from Deinococcus geothermalis, Geobacillus thermoglucosidasius and Aeropyrum pernix geothermalis were prepared and their thermostability, kinetic properties, substrate specificities towards (deoxy)adenosine, (deoxy)inosine, (deoxy)cytidine and C2′‐modified (aminodeoxy, deoxyfluoro) nucleosides investigated. DgPNP and ApMTAP are promising biocatalysts in the synthesis of 9‐(2‐deoxy‐2‐fluoro‐‐D‐ribo/arabino‐furanosyl)adenines using the corresponding uracil deoxyfluoro nucleosides as the pentofuranose donors in the transglycosylation reaction of adenine
Background Pernisine is an extracellular serine protease from the hyperthermophilic Archaeon Aeropyrum pernix K1. Low yields from the natural host and expression problems in heterologous hosts have ...limited the potential applications of pernisine in industry. Methodology/ Principal Findings The challenges of pernisine overexpression in Escherichia coli were overcome by codon preference optimisation and de-novo DNA synthesis. The following forms of the pernisine gene were cloned into the pMCSGx series of vectors and expressed in E. coli cells: wild-type (pernisinewt), codon-optimised (pernisineco), and codon-optimised with a S355A mutation of a predicted active site (pernisineS355Aco). The fusion-tagged pernisines were purified using fast protein liquid chromatography equipped with Ni2+ chelate and gel filtration chromatography columns. The identities of the resultant proteins were confirmed with N-terminal sequencing, tandem mass spectrometry analysis, and immunodetection. Pernisinewt was not expressed in E. coli at detectable levels, while pernisineco and pernisineS355Aco were expressed and purified as 55-kDa proforms with yields of around 10 mg per litre E. coli culture. After heat activation of purified pernisine, the proteolytic activity of the mature pernisineco was confirmed using zymography, at a molecular weight of 36 kDa, while the mutant pernisineS355Aco remained inactive. Enzymatic performances of pernisine evaluated under different temperatures and pHs demonstrate that the optimal enzymatic activity of the recombinant pernisine is ca. 100 degree C and pH 7.0, respectively. Conclusions/ Significance These data demonstrate that codon optimisation is crucial for pernisine overexpression in E. coli, and that the proposed catalytic Ser355 has an important role in pernisine activity, but not in its activation process. Pernisine is activated by autoproteolytical cleavage of its N-terminal proregion. We have also confirmed that the recombinant pernisine retains the characteristics of native pernisine, as a calcium modulated thermostable serine protease.
All organisms universally encode, synthesize and utilize proteins that function optimally within a subset of growth conditions. While healthy cells are thought to maintain high translational fidelity ...within their natural habitats, natural environments can easily fluctuate outside the optimal functional range of genetically encoded proteins. The hyperthermophilic archaeon Aeropyrum pernix (A. pernix) can grow throughout temperature variations ranging from 70 to 100°C, although the specific factors facilitating such adaptability are unknown. Here, we show that A. pernix undergoes constitutive leucine to methionine mistranslation at low growth temperatures. Low-temperature mistranslation is facilitated by the misacylation of tRNA(Leu) with methionine by the methionyl-tRNA synthetase (MetRS). At low growth temperatures, the A. pernix MetRS undergoes a temperature dependent shift in tRNA charging fidelity, allowing the enzyme to conditionally charge tRNA(Leu) with methionine. We demonstrate enhanced low-temperature activity for A. pernix citrate synthase that is synthesized during leucine to methionine mistranslation at low-temperature growth compared to its high-fidelity counterpart synthesized at high-temperature. Our results show that conditional leucine to methionine mistranslation can make protein adjustments capable of improving the low-temperature activity of hyperthermophilic proteins, likely by facilitating the increasing flexibility required for greater protein function at lower physiological temperatures.
Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. ...This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 A, and of the isolated voltage-sensor domain at 1.9 A, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.
Virus capsid proteins have various applications in diverse fields such as biotechnology, electronics, and medicine. In this study, the major capsid protein of bacilliform clavavitus APBV1, which ...infects the hyperthermophilic archaeon
Aeropyrum pernix
, was successfully expressed in
Escherichia coli
. The gene product was expressed as a histidine-tagged protein in
E. coli
and purified to homogeneity using single-step nickel affinity chromatography. The purified recombinant protein self-assembled to form bacilliform virus-like particles at room temperature. The particles exhibited tolerance against high concentrations of organic solvents and protein denaturants. In addition, we succeeded in fabricating functional nanoparticles with amine functional groups on the surface of ORF6-81 nanoparticles. These robust protein nanoparticles can potentially be used as a scaffold in nanotechnological applications.
The oxidation of thiol groups in proteins is a common event in biochemical processes involving disulfide bond formation and in response to an increased level of reactive oxygen species. It has been ...widely accepted that the oxidation of a cysteine side chain is initiated by the formation of cysteine sulfenic acid (Cys-SOH). Here, we demonstrate a mechanism of thiol oxidation through a hypervalent sulfur intermediate by presenting crystallographic evidence from an archaeal peroxiredoxin (Prx), the thioredoxin peroxidase from Aeropyrum pernix K1 (ApTPx). The reaction of Prx, which is the reduction of a peroxide, depends on the redox active cysteine side chains. Oxidation by hydrogen peroxide converted the active site peroxidatic Cys-50 of ApTPx to a cysteine sulfenic acid derivative, followed by further oxidation to cysteine sulfinic and sulfonic acids. The crystal structure of the cysteine sulfenic acid derivative was refined to 1.77 Å resolution with Rcryst and Rfree values of 18.8% and 22.0%, respectively. The refined structure, together with quantum chemical calculations, revealed that the sulfenic acid derivative is a type of sulfurane, a hypervalent sulfur compound, and that the Sγ atom is covalently linked to the Nδ¹ atom of the neighboring His-42. The reaction mechanism is revealed by the hydrogen bond network around the peroxidatic cysteine and the motion of the flexible loop covering the active site and by quantum chemical calculations. This study provides evidence that a hypervalent sulfur compound occupies an important position in biochemical processes.
Archaeal viruses have evolved to infect hosts often thriving in extreme conditions such as high temperatures. However, there is a paucity of information on archaeal virion structures, genome ...packaging, and determinants of temperature resistance. The rod-shaped virus APBV1 (Aeropyrum pernix bacilliform virus 1) is among the most thermostable viruses known; it infects a hyperthermophile Aeropyrum pernix, which grows optimally at 90 °C. Here we report the structure of APBV1, determined by cryo-electron microscopy at near-atomic resolution. Tight packing of the major virion glycoprotein (VP1) is ensured by extended hydrophobic interfaces, and likely contributes to the extreme thermostability of the helical capsid. The double-stranded DNA is tightly packed in the capsid as a left-handed superhelix and held in place by the interactions with positively charged residues of VP1. The assembly is closed by specific capping structures at either end, which we propose to play a role in DNA packing and delivery.
The quaternary structure of peroxiredoxin from Aeropyrum pernix K1 (ApPrx) is a decamer, in which five homodimers are assembled in a pentagonal ring through hydrophobic interactions. In this study, ...we determined the amino acid (AA) residues of ApPrx crucial for forming the decamer using AA mutations. The ApPrx0Cys mutant, wherein all cysteine residues were mutated to serine, was prepared as a model protein to remove the influence of the redox states of the cysteines on its assembling behavior. The boundary between each homodimer of ApPrx0Cys contains characteristic aromatic AA residues forming hydrophobic interactions: F46, F80, W88, W210, and W211. We found that a single mutation of F46, F80, or W210 to alanine completely disassembled the ApPrx0Cys decamer to homodimers, which was clarified by gel‐filtration chromatography and dynamic light scattering measurements. F46A, F80A, and W210A mutants lacked only one aromatic ring compared with ApPrx0Cys, indicating that the assembly is very sensitive to the surface structure of the protein. X‐ray structures revealed two mechanisms of disassembly of the ApPrx decamer: loss of hydrophobicity between homodimers and flip of the arm domain. The AA residues targeted in this study are well conserved in ring‐type Prx proteins, suggesting the importance of these residues in the assembly. This study demonstrates the sensitivity and modifiability of peroxiredoxin assembly by a simple AA mutation.
Molecular dynamics (MD) simulations were used to investigate the electroporation of archaeal lipid bilayers when subjected to high transmembrane voltages induced by a charge imbalance, mimicking ...therefore millisecond electric pulse experiments. The structural characteristics of the bilayer, a 9:91mol% 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-myo-inositol (AI) and 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-1′(2′-O-α-d-glucosyl)-myo-inositol (AGI) were compared to small angle X-ray scattering data. A rather good agreement of the electron density profiles at temperatures of 298 and 343K was found assessing therefore the validity of the protocols and force fields used in simulations. Compared to dipalmitoyl-phosphatidylcholine (DPPC), the electroporation threshold for the bilayer was found to increase from ~2V to 4.3V at 323K, and to 5.2V at 298K. Comparing the electroporation thresholds of the archaeal lipids to those of simple diphytanoyl-phosphatidylcholine (DPhPC) bilayers (2.5V at 323K) allowed one to trace back the stability of the membranes to the structure of their lipid head groups. Addition of DPPC in amounts of 50mol% to the archaeal lipid bilayers decreases their stability and lowers the electroporation thresholds to 3.8V and 4.1V at respectively 323 and 298K. The present study therefore shows how membrane compositions can be selected to cover a wide range of responses to electric stimuli. This provides new routes for the design of liposomes that can be efficiently used as drug delivery carriers, as the selection of their composition allows one to tune in their electroporation threshold for subsequent release of their load.
•MD simulations are in good agreement with SAXS results.•The archaeal lipid bilayers have high electroporation threshold.•During electroporation in archaeal lipid bilayers, we observed hydrophobic pores.
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