L-Asparaginase Conjugates from the Hyperthermophilic Archaea Thermococcus sibiricus with Improved Biocatalytic Properties.

This study investigated the effect of polycationic and uncharged polymers (and oligomers) on the catalytic parameters and thermostability of L-asparaginase from Thermococcus sibiricus (TsA). This enzyme has potential applications in the food industry to decrease the formation of carcinogenic acrylamide during the processing of carbohydrate-containing products. Conjugation with the polyamines polyethylenimine and spermine (PEI and Spm) or polyethylene glycol (PEG) did not significantly affect the secondary structure of the enzyme. PEG contributes to the stabilization of the dimeric form of TsA, as shown by HPLC. Furthermore, neither polyamines nor PEG significantly affected the binding of the L-Asn substrate to TsA. The conjugates showed greater maximum activity at pH 7.5 and 85 °C, 10-50% more than for native TsA. The pH optima for both TsA-PEI and TsA-Spm conjugates were shifted to lower pH ranges from pH 10 (for the native enzyme) to pH 8.0. Additionally, the TsA-Spm conjugate exhibited the highest activity at pH 6.5-9.0 among all the samples. Furthermore, the temperature optimum for activity at pH 7.5 shifted from 90-95 °C to 80-85 °C for the conjugates. The thermal inactivation mechanism of TsA-PEG appeared to change, and no aggregation was observed in contrast to that of the native enzyme. This was visually confirmed and supported by the analysis of the CD spectra, which remained almost unchanged after heating the conjugate solution. These results suggest that TsA-PEG may be a more stable form of TsA, making it a potentially more suitable option for industrial use.

Crystal structure of the 3'→5' exonuclease from Methanocaldococcus jannaschii.

RecJ exonucleases are members of the DHH phosphodiesterase family ancestors of eukaryotic Cdc45, the key component of the CMG (Cdc45-MCM-GINS) complex at the replication fork. They are involved in DNA replication and repair, RNA maturation and Okazaki fragment degradation. Bacterial RecJs resect 5'-end ssDNA. Conversely, archaeal RecJs are more versatile being able to hydrolyse in both directions and acting on ssDNA as well as on RNA. In Methanocaldococcus jannaschii two RecJs were previously characterized: RecJ1 is a 5'→3' DNA exonuclease, MjaRecJ2 works only on 3'-end DNA/RNA with a preference for RNA. Here, I present the crystal structure of MjaRecJ2, solved at a resolution of 2.8 Å, compare it with the other RecJ structures, in particular the 5'→3' TkoGAN and the bidirectional PfuRecJ, and discuss its characteristics in light of the more recent knowledge on RecJs. This work adds new structural data that might improve the knowledge of these class of proteins.

Kinetic, electrochemical and spectral characterization of bacterial and archaeal rusticyanins; unexpected stability issues and consequences for applications in biotechnology.

Motivated by the ambition to establish an enzyme-driven bioleaching pathway for copper extraction, properties of the Type-1 copper protein rusticyanin from Acidithiobacillus ferrooxidans (AfR) were compared with those from an ancestral form of this enzyme (N0) and an archaeal enzyme identified in Ferroplasma acidiphilum (FaR). While both N0 and FaR show redox potentials similar to that of AfR their electron transport rates were significantly slower. The lack of a correlation between the redox potentials and electron transfer rates indicates that AfR and its associated electron transfer chain evolved to specifically facilitate the efficient conversion of the energy of iron oxidation to ATP formation. In F. acidiphilum this pathway is not as efficient unless it is up-regulated by an as of yet unknown mechanism. In addition, while the electrochemical properties of AfR were consistent with previous data, previously unreported behavior was found leading to a form that is associated with a partially unfolded form of the protein. The cyclic voltammetry (CV) response of AfR immobilized onto an electrode showed limited stability, which may be connected to the presence of the partially unfolded state of this protein. Insights gained in this study may thus inform the engineering of optimized rusticyanin variants for bioleaching processes as well as enzyme-catalyzed solubilization of copper-containing ores such as chalcopyrite.

High-resolution crystal structure of RNA kinase ArK1 from G. acetivorans.

U47 phosphorylation (Up47) is a novel tRNA modification discovered recently; it can confer thermal stability and nuclease resistance to tRNAs. U47 phosphorylation is catalyzed by Archaeal RNA kinase (Ark1) in an ATP-dependent manner. However, the structural basis for tRNA and/or ATP binding by Ark1 is unclear. Here, we report the expression, purification, and crystallization studies of Ark1 from G. acetivorans (GaArk1). In addition to the Apo-form structure, one GaArk1-ATP complex was also determined in atomic resolution and revealed the detailed basis for ATP binding by GaArk1. The GaArk1-ATP complex represents the only ATP-bound structure of the Ark1 protein. The majority of the ATP-binding residues are conserved, suggesting that GaArk1 and the homologous proteins share similar mechanism in ATP binding. Sequence and structural analysis further indicated that endogenous guanosine will only inhibit the activities of certain Ark1 proteins, such as Ark1 from T. kodakarensis.

Characterisation and engineering of a thermophilic RNA ligase from Palaeococcus pacificus.

RNA ligases are important enzymes in molecular biology and are highly useful for the manipulation and analysis of nucleic acids, including adapter ligation in next-generation sequencing of microRNAs. Thermophilic RNA ligases belonging to the RNA ligase 3 family are gaining attention for their use in molecular biology, for example a thermophilic RNA ligase from Methanobacterium thermoautotrophicum is commercially available for the adenylation of nucleic acids. Here we extensively characterise a newly identified RNA ligase from the thermophilic archaeon Palaeococcus pacificus (PpaRnl). PpaRnl exhibited significant substrate adenylation activity but low ligation activity across a range of oligonucleotide substrates. Mutation of Lys92 in motif I to alanine, resulted in an enzyme that lacked adenylation activity, but demonstrated improved ligation activity with pre-adenylated substrates (ATP-independent ligation). Subsequent structural characterisation revealed that in this mutant enzyme Lys238 was found in two alternate positions for coordination of the phosphate tail of ATP. In contrast mutation of Lys238 in motif V to glycine via structure-guided engineering enhanced ATP-dependent ligation activity via an arginine residue compensating for the absence of Lys238. Ligation activity for both mutations was higher than the wild-type, with activity observed across a range of oligonucleotide substrates with varying sequence and secondary structure.

Crystal structure of GTP-dependent dephospho-coenzyme A kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis.

The biosynthesis pathways of coenzyme A (CoA) in most archaea involve several unique enzymes including dephospho-CoA kinase (DPCK) that converts dephospho-CoA to CoA in the final step of CoA biosynthesis in all domains of life. The archaeal DPCK is unrelated to the analogous bacterial and eukaryotic enzymes and shows no significant sequence similarity to any proteins with known structures. Unusually, the archaeal DPCK utilizes GTP as the phosphate donor although the analogous bacterial and eukaryotic enzymes are ATP-dependent kinases. Here, we report the crystal structure of DPCK and its complex with GTP and a magnesium ion from the archaeal hyperthermophile Thermococcus kodakarensis. The crystal structure demonstrates why GTP is the preferred substrate of this kinase. We also report the activity analyses of site-directed mutants of crucial residues determined based on sequence conservation and the crystal structure. From these results, the key residues involved in the reaction of phosphoryl transfer and the possible dephospho-CoA binding site are inferred.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

The archaeal Lsm protein from Pyrococcus furiosus binds co-transcriptionally to poly(U)-rich target RNAs.

Posttranscriptional processes in Bacteria include the association of small regulatory RNAs (sRNA) with a target mRNA. The sRNA/mRNA annealing process is often mediated by an RNA chaperone called Hfq. The functional role of bacterial and eukaryotic Lsm proteins is partially understood, whereas knowledge about archaeal Lsm proteins is scarce. Here, we used the genetically tractable archaeal hyperthermophile Pyrococcus furiosus to identify the protein interaction partners of the archaeal Sm-like proteins (PfuSmAP1) using mass spectrometry and performed a transcriptome-wide binding site analysis of PfuSmAP1. Most of the protein interaction partners we found are part of the RNA homoeostasis network in Archaea including ribosomal proteins, the exosome, RNA-modifying enzymes, but also RNA polymerase subunits, and transcription factors. We show that PfuSmAP1 preferentially binds messenger RNAs and antisense RNAs recognizing a gapped poly(U) sequence with high affinity. Furthermore, we found that SmAP1 co-transcriptionally associates with target RNAs. Our study reveals that in contrast to bacterial Hfq, PfuSmAP1 does not affect the transcriptional activity or the pausing behaviour of archaeal RNA polymerases. We propose that PfuSmAP1 recruits antisense RNAs to target mRNAs and thereby executes its putative regulatory function on the posttranscriptional level.

Archaella Isolation.

Swimming archaea are propelled by a filamentous structure called the archaellum. The first step for the structural characterization of this filament is its isolation. Here we provide various methods that allow for the isolation of archaella filaments from well-studied archaeal model organisms. Archaella filaments have been successfully extracted from organisms belonging to different archaeal phyla, e.g., euryarchaeal methanogens such as Methanococcus voltae, and crenarchaeal hyperthermoacidophiles like Sulfolobus acidocaldarius. The filament isolation protocols that we provide in this chapter follow one of two strategies: either the filaments are sheared or extracted from whole cells by detergent extraction, prior to further final purification by centrifugation methods.

Evolution and Diversity of the TopoVI and TopoVI-like Subunits With Extensive Divergence of the TOPOVIBL subunit.

Type II DNA topoisomerases regulate topology by double-stranded DNA cleavage and ligation. The TopoVI family of DNA topoisomerase, first identified and biochemically characterized in Archaea, represents, with TopoVIII and mini-A, the type IIB family. TopoVI has several intriguing features in terms of function and evolution. TopoVI has been identified in some eukaryotes, and a global view is lacking to understand its evolutionary pattern. In addition, in eukaryotes, the two TopoVI subunits (TopoVIA and TopoVIB) have been duplicated and have evolved to give rise to Spo11 and TopoVIBL, forming TopoVI-like (TopoVIL), a complex essential for generating DNA breaks that initiate homologous recombination during meiosis. TopoVIL is essential for sexual reproduction. How the TopoVI subunits have evolved to ensure this meiotic function is unclear. Here, we investigated the phylogenetic conservation of TopoVI and TopoVIL. We demonstrate that BIN4 and RHL1, potentially interacting with TopoVIB, have co-evolved with TopoVI. Based on model structures, this observation supports the hypothesis for a role of TopoVI in decatenation of replicated chromatids and predicts that in eukaryotes the TopoVI catalytic complex includes BIN4 and RHL1. For TopoVIL, the phylogenetic analysis of Spo11, which is highly conserved among Eukarya, highlighted a eukaryal-specific N-terminal domain that may be important for its regulation. Conversely, TopoVIBL was poorly conserved, giving rise to ATP hydrolysis-mutated or -truncated protein variants, or was undetected in some species. This remarkable plasticity of TopoVIBL provides important information for the activity and function of TopoVIL during meiosis.

Prokaryotic innate immunity through pattern recognition of conserved viral proteins.

Many organisms have evolved specialized immune pattern-recognition receptors, including nucleotide-binding oligomerization domain-like receptors (NLRs) of the STAND superfamily that are ubiquitous in plants, animals, and fungi. Although the roles of NLRs in eukaryotic immunity are well established, it is unknown whether prokaryotes use similar defense mechanisms. Here, we show that antiviral STAND (Avs) homologs in bacteria and archaea detect hallmark viral proteins, triggering Avs tetramerization and the activation of diverse N-terminal effector domains, including DNA endonucleases, to abrogate infection. Cryo-electron microscopy reveals that Avs sensor domains recognize conserved folds, active-site residues, and enzyme ligands, allowing a single Avs receptor to detect a wide variety of viruses. These findings extend the paradigm of pattern recognition of pathogen-specific proteins across all three domains of life.

The structure and activities of the archaeal transcription termination factor Eta detail vulnerabilities of the transcription elongation complex.

Transcription must be properly regulated to ensure dynamic gene expression underlying growth, development, and response to environmental cues. Regulation is imposed throughout the transcription cycle, and while many efforts have detailed the regulation of transcription initiation and early elongation, the termination phase of transcription also plays critical roles in regulating gene expression. Transcription termination can be driven by only a few proteins in each domain of life. Detailing the mechanism(s) employed provides insight into the vulnerabilities of transcription elongation complexes (TECs) that permit regulated termination to control expression of many genes and operons. Here, we describe the biochemical activities and crystal structure of the superfamily 2 helicase Eta, one of two known factors capable of disrupting archaeal transcription elongation complexes. Eta retains a twin-translocase core domain common to all superfamily 2 helicases and a well-conserved C terminus wherein individual amino acid substitutions can critically abrogate termination activities. Eta variants that perturb ATPase, helicase, single-stranded DNA and double-stranded DNA translocase and termination activities identify key regions of the C terminus of Eta that, when combined with modeling Eta-TEC interactions, provide a structural model of Eta-mediated termination guided in part by structures of Mfd and the bacterial TEC. The susceptibility of TECs to disruption by termination factors that target the upstream surface of RNA polymerase and potentially drive termination through forward translocation and allosteric mechanisms that favor opening of the clamp to release the encapsulated nucleic acids emerges as a common feature of transcription termination mechanisms.

In Vitro Transcription Assay for Archaea Belonging to Sulfolobales.

Archaeal transcription and its regulation are characterized by a mosaic of eukaryotic and bacterial features. Molecular analysis of the functioning of the archaeal RNA polymerase, basal transcription factors, and specific promoter-containing DNA templates allows to unravel the mechanisms of transcription regulation in archaea. In vitro transcription is a technique that allows the study of this process in a simplified and controlled environment less complex than the archaeal cell. In this chapter, we present an in vitro transcription methodology for the study of transcription in Sulfolobales. It is described how to purify the RNA polymerase and the basal transcription factors TATA-binding protein and transcription factor B of Saccharolobus solfataricus and how to perform in vitro transcription reactions and transcript detection. Application of this protocol for other archaeal species could require minor modifications to protein overexpression and purification conditions.

Discovery, structure and mechanism of a tetraether lipid synthase.

Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1-5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon-carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon-carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3-Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7-10.

Mechanisms of helicase activated DNA end resection in bacteria.

DNA end resection mediated by the coordinated action of nuclease and helicase is a crucial step in initiating homologous recombination. The end-resection apparatus NurA nuclease and HerA helicase are present in both archaea and bacteria. Here, we report the cryo-electron microscopy structure of a bacterial HerA-NurA complex from Deinococcus radiodurans. The structure reveals a barrel-like hexameric HerA and a distinctive NurA dimer subcomplex, which has a unique extended N-terminal region (ENR) involved in bacterial NurA dimerization and activation. In addition to the long protruding linking loop and the C-terminal α helix of NurA, the flexible ENR is close to the HerA-NurA interface and divides the central channel of the DrNurA dimer into two halves, suggesting a possible mechanism of DNA end processing. In summary, this work provides new insights into the structure, assembly, and activation mechanisms of bacterial DNA end resection mediated by a minimal end-resection apparatus.

Archaeal bundling pili of Pyrobaculum calidifontis reveal similarities between archaeal and bacterial biofilms.

While biofilms formed by bacteria have received great attention due to their importance in pathogenesis, much less research has been focused on the biofilms formed by archaea. It has been known that extracellular filaments in archaea, such as type IV pili, hami, and cannulae, play a part in the formation of archaeal biofilms. We have used cryo-electron microscopy to determine the atomic structure of a previously uncharacterized class of archaeal surface filaments from hyperthermophilic Pyrobaculum calidifontis. These filaments, which we call archaeal bundling pili (ABP), assemble into highly ordered bipolar bundles. The bipolar nature of these bundles most likely arises from the association of filaments from at least two different cells. The component protein, AbpA, shows homology, both at the sequence and structural level, to the bacterial protein TasA, a major component of the extracellular matrix in bacterial biofilms, contributing to biofilm stability. We show that AbpA forms very stable filaments in a manner similar to the donor-strand exchange of bacterial TasA fibers and chaperone-usher pathway pili where a ß-strand from one subunit is incorporated into a ß-sheet of the next subunit. Our results reveal likely mechanistic similarities and evolutionary connection between bacterial and archaeal biofilms, and suggest that there could be many other archaeal surface filaments that are as yet uncharacterized.

Essentiality of core hydrophobicity to the structure and function of archaeal chromatin protein Cren7.

Studies on the structure-function relationship of protein greatly help to understand not only the principles of protein folding but also the rationales of protein engineering. Crenarchaeal chromatin protein Cren7 provides an excellent research model for this issue. The small protein adopts a 'ß-barrel' fold, formed by the double-stranded antiparallel ß-sheet 1 tightly packing with the triple-stranded antiparallel ß-sheet 2. The simple structure of Cren7 is stabilized by the hydrophobic core between the ß-sheets, consisting of the side chains of V8, V10, L20, V25, F41 and F50. In the present work, mutation analyses by alanine substitution of each of the residues in the hydrophobic core were performed. Circular dichroism spectra and nuclear magnetic resonance analyses showed that mutation of F41 led to a significant misfolding of Cren7 through disruption of the ß-sheets. Meanwhile, the mutant F41A showed a reduced thermostatility (Tm of 53.2 °C), as compared with the wild-type Cren7 (Tm > 80 °C). Biolayer interferometry and nick-closure assays showed the largely unchanged activities in DNA binding and supercoiling of F41A, indicating the DNA interface of Cren7 was generally retained in F41A. However, F41A was unable to mediate DNA bridging, probably due to the impairment in forming oligomers/polymers on DNA. Atomic force microscopic images of the F41A-DNA complexes also revealed that F41A nearly completely lost the ability to compact DNA into highly condensed structures. Our results not only reveal the critical role of F41 in protein folding of Cren7 but also provide new insights into the structure-function relationships of thermostable proteins.

Machine learning and statistics shape a novel path in archaeal promoter annotation.

BACKGROUND: Archaea are a vast and unexplored domain. Bioinformatic techniques might enlighten the path to a higher quality genome annotation in varied organisms. Promoter sequences of archaea have the action of a plethora of proteins upon it. The conservation found in a structural level of the binding site of proteins such as TBP, TFB, and TFE aids RNAP-DNA stabilization and makes the archaeal promoter prone to be explored by statistical and machine learning techniques. RESULTS AND DISCUSSIONS: In this study, experimentally verified promoter sequences of the organisms Haloferax volcanii, Sulfolobus solfataricus, and Thermococcus kodakarensis were converted into DNA duplex stability attributes (i.e. numerical variables) and were classified through Artificial Neural Networks and an in-house statistical method of classification, being tested with three forms of controls. The recognition of these promoters enabled its use to validate unannotated promoter sequences in other organisms. As a result, the binding site of basal transcription factors was located through a DNA duplex stability codification. Additionally, the classification presented satisfactory results (above 90%) among varied levels of control. CONCLUDING REMARKS: The classification models were employed to perform genomic annotation into the archaea Aciduliprofundum boonei and Thermofilum pendens, from which potential promoters have been identified and uploaded into public repositories.

Structural Characterization of a Thorarchaeota Profilin Indicates Eukaryotic-Like Features but with an Extended N-Terminus.

The emergence of the first eukaryotic cell is preceded by evolutionary events, which are still highly debatable. Clues of the exact sequence of events are beginning to emerge. Recent metagenomics analyses has uncovered the Asgard super-phylum as the closest yet known archaea host of eukaryotes. Some of these have been tested and confirmed experimentally. However, the bulk of eukaryotic signature proteins predicted to be encoded by the Asgard super-phylum have not been studied, and their true functions, at least in the context of a eukaryotic cell, are still elusive. For example, there are several different variants of the profilin within each Asgardian Achaea, and there are some conflicting results of their actual roles. Here, the 3D structure of profilin from Thorarchaeota is determined by nuclear magnetic resonance spectroscopy and shows that this profilin has a eukaryotic-like profilin with a rigid core and an extended N-terminus previously implicated in polyproline binding. In addition, it is also shown that Thorarchaeota Profilin co-localizes with eukaryotic actin in cultured HeLa cells. This finding reaffirms the notion that Asgardian encoded proteins possess eukaryotic-like characteristics and strengthen the likely existence of a complex cytoskeleton already in a last eukaryotic common ancestor.

Molecular cloning, expression in Escherichia coli and structural-functional analysis of a pyruvate kinase from Pyrobaculum calidifontis.

This manuscript describes recombinant production, characterization and structural analysis of wild-type and mutant Pcal_0029, a pyruvate kinase from Pyrobaculum calidifontis. Recombinant Pcal_0029 was produced in soluble and highly active form in Escherichia coli. Purified protein exhibited divalent metal-dependent activity which increased with the increase in temperature till 85 °C. Recombinant Pcal_0029 was highly thermostable with no significant loss in activity even after an incubation of 120 min at 100 °C. The enzyme exhibited apparent S0.5 and Vmax values of 0.44 ± 0.05 mM and 840 ± 39 units, respectively, towards phosphoenolpyruvate. These values towards adenosine-5'-diphosphate were 0.5 ± 0.07 mM and 870 ± 26 units, respectively. In silico structural analysis and comparison with the characterized enzymes revealed the presence of eight conserved regions. Two substitutions, K130E and S155G, resulted in a 10-fold decrease in activity. Secondary structure analysis indicated similar structures for the wild-type and the mutant enzymes. Bioinformatics analysis revealed disruption of interatomic interactions and hydrogen bonds, leading to a decreased flexibility and solvent accessibility, which may have led to decrease in activity. To the best of our knowledge, Pcal_0029 is the most thermostable pyruvate kinase reported so far. Moreover, this is the first study on the role of non-catalytic residues in a pyruvate kinase.