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High fidelity DNA polymerase from Pyrococcus furiosus (Pfupol) is an attractive alternative to the highly popular DNA polymerase from Thermus aquaticus. Because this enzyme is in great demand for biotechnological applications, optimizing Pfupol production is essential to supplying the industry's expanding demand. T7-induced promoter expression in Escherichia coli expression systems is used to express recombinant Pfupol; however, this method is not cost-effective. Here, we have effectively developed an optimized process for the autoinduction approach of Pfupol expression in a defined medium. To better examine Pfupol's activities, its purified fraction was used. A 71 mg/L of pure Pfupol was effectively produced, resulting in a 2.6-fold increase in protein yield when glucose, glycerol, and lactose were added in a defined medium at concentrations of 0.05%, 1%, and 0.6%, respectively, and the condition for production in a 5 L bioreactor was as follow: 200 rpm, 3 vvm, and 10% inoculant. Furthermore, the protein exhibited 1445 U/mg of specific activity when synthesized in its active state. This work presents a high level of Pfupol production, which makes it an economically viable and practically useful approach., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

This study introduces an efficient RPA-PfAgo detection system for the MTHFR C677T polymorphism, proposing a potential strategy to simplify the genotyping process. By optimizing recombinase polymerase amplification (RPA) with Pyrococcus furiosus Argonaute (PfAgo) nucleases, we achieved DNA amplification at a constant temperature. The assay was fine-tuned through meticulous primer and guide DNA selection, with optimal conditions established at 2.0 µL of MgAc, a reaction temperature of 42 °C, and a 10-minute reaction time for RPA. Further optimization of the PfAgo cleavage assay revealed the ideal concentrations of MnCl 2 , guide DNA, molecular beacon probes, the PfAgo enzyme, and the RPA product to maximize sensitivity and specificity. Clinical validation of 20 samples showed 100% concordance with Sanger sequencing, confirming the method's precision. The RPA-PfAgo system is a promising tool for on-site genotyping, with broad applications in personalized medicine and disease prevention., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric β-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1., (© 2024. The Author(s).)

Foodborne hazardous factors pose a significant risk to public health, emphasizing the need for the development of sensitive and user-friendly detection strategies to effectively manage and control these risks in the food supply chain. Pyrococcus furiosus argonaute (PfAgo)-based biosensing approaches have been extensively explored due to its built-in signal amplification. However, the property that PfAgo is a DNA-guided DNA endonuclease has enabled almost all the existing PfAgo-based reports to be used for the detection of nucleic acids. To lend PfAgo toolbox to extended non-nucleic acid detection, we systematically investigated the mechanism characteristic of PfAgo' preference for guide DNA (gDNA) and proposed a gDNA dephosphorylation-modulated PfAgo sensor for the detection of non-nucleic acid targets. Our results indicated that PfAgo exhibits preference for 5'-phosphorylated gDNA at a specific ratio of PfAgo to gDNA concentration. Leveraging this PfAgo' preference and the dephosphorylation activity of alkaline phosphatase (ALP), ALP could be detected as low as 2.7 U/L. Furthermore, the PfAgo was coupled with immunolabelled ALP to develop a PfAgo-based fluorescence immunosensor, which achieves aflatoxins B 1 detection with a detection limit of 29.89 pg/mL and exhibits satisfactory recoveries in wheat and maize samples. The developed method broadens the application scope of PfAgo toolbox, and provides a simple, sensitive, and universal detection platform for a variety targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hyperthermophilic archaea such as Pyrococcus furiosus survive under very aggressive environmental conditions by occupying niches inaccessible to representatives of other domains of life. The ability to survive such severe living conditions must be ensured by extraordinarily efficient mechanisms of DNA processing, including repair. Therefore, in this study, we compared kinetics of conformational changes of DNA Endonuclease Q from P. furiosus during its interaction with various DNA substrates containing an analog of an apurinic/apyrimidinic site (F-site), hypoxanthine, uracil, 5,6-dihydrouracil, the α-anomer of adenosine, or 1, N 6 -ethenoadenosine. Our examination of DNA cleavage activity and fluorescence time courses characterizing conformational changes of the dye-labeled DNA substrates during the interaction with EndoQ revealed that the enzyme induces multiple conformational changes of DNA in the course of binding. Moreover, the obtained data suggested that the formation of the enzyme-substrate complex can proceed through dissimilar kinetic pathways, resulting in different types of DNA conformational changes, which probably allow the enzyme to perform its biological function at an extreme temperature.

The membrane-bound hydrogenase (Mbh) from Pyrococcus furiosus is an archaeal member of the Complex I superfamily. It catalyzes the reduction of protons to H 2 gas powered by a [NiFe] active site and transduces the free energy into proton pumping and Na + /H + exchange across the membrane. Despite recent structural advances, the mechanistic principles of H 2 catalysis and ion transport in Mbh remain elusive. Here, we probe how the redox chemistry drives the reduction of the proton to H 2 and how the catalysis couples to conformational dynamics in the membrane domain of Mbh. By combining large-scale quantum chemical density functional theory (DFT) and correlated ab initio wave function methods with atomistic molecular dynamics simulations, we show that the proton transfer reactions required for the catalysis are gated by electric field effects that direct the protons by water-mediated reactions from Glu21 L toward the [NiFe] site, or alternatively along the nearby His75 L pathway that also becomes energetically feasible in certain reaction steps. These local proton-coupled electron transfer (PCET) reactions induce conformational changes around the active site that provide a key coupling element via conserved loop structures to the ion transport activity. We find that H 2 forms in a heterolytic proton reduction step, with spin crossovers tuning the energetics along key reaction steps. On a general level, our work showcases the role of electric fields in enzyme catalysis and how these effects are employed by the [NiFe] active site of Mbh to drive PCET reactions and ion transport.

A Pyrococcus furiosus Argonaute (PfAgo)-based biosensor is presented for alkaline phosphatase (ALP) activity detection in which the ALP-catalyzed hydrolysis of 3'-phosphate-modified functional DNA activates the strand displacement amplification, and the amplicon mediates the fluorescent reporter cleavage as a guide sequence of PfAgo. Under the dual amplification mode of PfAgo-catalyzed multiple-turnover cleavage activity and pre-amplification technology, the developed method was successfully applied to ALP activity determination with a detection limit (LOD) of 0.0013 U L -1 (3σ) and a detection range of 0.0025 to 1 U L -1 within 90 min. The PfAgo-based method exhibits satisfactory analytic performance in the presence of potential interferents and in complex human serum samples. The proposed method shows several advantages, such as rapid analysis, high sensitivity, low-cost, and easy operation, and has great potential in disease evolution fundamental studies and clinical diagnosis applications., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Hyperthermophilic enzymes serve as an important source of industrial enzymes due to their high thermostability. Unfortunately, most hyperthermophilic enzymes suffer from reduced activity at low temperatures (e.g., ambient temperature), limiting their applicability. In addition, evolving hyperthermophilic enzymes to increase low temperature activity without compromising other desired properties is generally difficult. In the current study, a variant of β-glucosidase from Pyrococcus furiosus (PfBGL) was engineered to enhance enzyme activity at low temperatures through the construction of a saturation mutagenesis library guided by the HotSpot Wizard analysis, followed by its screening for activity and thermostability. From this library construction and screening, one PfBGL mutant, PfBGL-A4 containing Q214S/A264S/F344I mutations, showed an over twofold increase in β-glucosidase activity at 25 and 50°C compared to the wild type, without compromising high-temperature activity, thermostability and substrate specificity. Our experimental and computational characterizations suggest that the findings with PfBGL-A4 may be due to the elevation of local conformational flexibility around the active site, while slightly compacting the global protein structure. This study showcases the potential of HotSpot Wizard-informed engineering of hyperthermophilic enzymes and underscores the interplays among temperature, enzyme activity, and conformational flexibility in these enzymes., (© 2024 Wiley Periodicals LLC.)

Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Crystal structure of a ribonuclease for ribosomal RNA processing, FAU-1, from Pyrococcus furiosus was determined with the resolution of 2.57 Å in a homo-trimeric form. The monomer structure consists of two domains: N-terminal and C-terminal domains. C-terminal domain forms trimer and each N-terminal domain locates outside of the trimer core. In the obtained crystal, a dinucleotide, pApUp, was bound to the N-terminal domain, indicating that N-terminal domain has the RNA-binding ability. The affinities to RNA of FAU-1 and a fragment corresponding to the N-terminal domain, FAU-ΔC, were confirmed by polyacrylamide gel electrophoresis and nuclear magnetic resonance (NMR). Interestingly, well-dispersed NMR signals were observed at 318K, indicating that the FAU-ΔC-F18 complex form an ordered structure at higher temperature. As predicted in our previous works, FAU-1 and ribonuclease (RNase) E show a structural similarity in their RNA-binding regions. However, structural similarity between RNase E and FAU-1 could be found in the limited regions of the N-terminal domain. On the other hand, structural similarity between C-terminal domain and some proteins including a phosphatase was found. Thus, it is possible that the catalytic site is located in C-terminal domain., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We have investigated the kinetics of NAD + -dependent NADPH:ferredoxin oxidoreductase (NfnI), a bifurcating transhydrogenase that takes two electron pairs from NADPH to reduce two ferredoxins and one NAD +  through successive bifurcation events. NADPH reduction takes place at the bifurcating FAD of NfnI's large subunit, with high-potential electrons transferred to the [2Fe-2S] cluster and S-FADH of the small subunit, ultimately on to NAD + ; low-potential electrons are transferred to two [4Fe-4S] clusters of the large subunit and on to ferredoxin. Reduction of NfnI by NADPH goes to completion only at higher pH, with a limiting k red  of 36 ± 1.6 s -1  and apparent K d NADPH  of 5 ± 1.2 μM. Reduction of one of the [4Fe-4S] clusters of NfnI occurs within a second, indicating that in the absence of NAD + , the system can bifurcate and generate low-potential electrons without NAD + . When enzyme is reduced by NADPH in the absence of NAD +  but the presence of ferredoxin, up to three equivalents of ferredoxin become reduced, although the reaction is considerably slower than seen during steady-state turnover. Bifurcation appears to be limited by transfer of the first, high-potential electron into the high-potential pathway. Ferredoxin reduction without NAD +  demonstrates that electron bifurcation is an intrinsic property of the bifurcating FAD and is not dependent on the simultaneous presence of NAD +  and ferredoxin. The tight coupling between NAD +  and ferredoxin reduction observed under multiple-turnover conditions is instead simply due to the need to remove reducing equivalents from the high-potential electron pathway under multiple-turnover conditions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

5'-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5'-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The membrane-bound hydrogenase (Mbh) is a redox-driven Na + /H + transporter that employs the energy from hydrogen gas (H 2 ) production to catalyze proton pumping and Na + /H + exchange across cytoplasmic membranes of archaea. Despite a recently resolved structure of this ancient energy-transducing enzyme [Yu et al. Cell 2018 , 173 , 1636-1649], the molecular principles of its redox-driven ion-transport mechanism remain puzzling and of major interest for understanding bioenergetic principles of early cells. Here we use atomistic molecular dynamics (MD) simulations in combination with data clustering methods and quantum chemical calculations to probe principles underlying proton reduction as well as proton and sodium transport in Mbh from the hyperthermophilic archaeon Pyrococcus furiosus . We identify putative Na + binding sites and proton pathways leading across the membrane and to the NiFe-active center as well as conformational changes that regulate ion uptake. We suggest that Na + binding and protonation changes at a putative ion-binding site couple to proton transfer across the antiporter-like MbhH subunit by modulating the conformational state of a conserved ion pair at the subunit interface. Our findings illustrate conserved coupling principles within the complex I superfamily and provide functional insight into archaeal energy transduction mechanisms.

Objective: With the widespread application of CRISPR/Cas9 gene editing technology, new methods are needed to screen mutants quickly and effectively. Here, we aimed to develop a simple and cost-effective method to screen CRISPR/Cas9-induced mutants., Result: We report a novel method to identify CRISPR/Cas9-induced mutants through a DNA-guided Argonaute nuclease derived from the archaeon Pyrococcus furiosus. We demonstrated that the Pyrococcus furiosus Argonaute (PfAgo)-based method could distinguish among biallelic mutants, monoallelic mutants and wild type (WT). Furthermore, this method was able to identify 1 bp indel mutations., Conclusion: The PfAgo-based method is simple to implement and can be applied to screen biallelic mutants and mosaic mutants generated by CRISPR-Cas9 or other kinds of gene editing tools., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We describe an approach for integrating distance restraints from Double Electron-Electron Resonance (DEER) spectroscopy into Rosetta with the purpose of modeling alternative protein conformations from an initial experimental structure. Fundamental to this approach is a multilateration algorithm that harnesses sets of interconnected spin label pairs to identify optimal rotamer ensembles at each residue that fit the DEER decay in the time domain. Benchmarked relative to data analysis packages, the algorithm yields comparable distance distributions with the advantage that fitting the DEER decay and rotamer ensemble optimization are coupled. We demonstrate this approach by modeling the protonation-dependent transition of the multidrug transporter PfMATE to an inward facing conformation with a deviation to the experimental structure of less than 2Å Cα RMSD. By decreasing spin label rotamer entropy, this approach engenders more accurate Rosetta models that are also more closely clustered, thus setting the stage for more robust modeling of protein conformational changes., Competing Interests: The authors have declared that no competing interests exist.

Salt-bridges play a key role in the thermostability of proteins adapted in stress environments whose intrinsic basis remains to be understood. We find that the higher hydrophilicity of PfP than that of HuP is due to the charged but not the polar residues. The primary role of these residues is to enhance the salt-bridges and their ME. Unlike HuP, PfP has made many changes in its intrinsic property to strengthen the salt-bridge. First, the desolvation energy is reduced by directing the salt-bridge towards the surface. Second, it has made bridge-energy more favorable by recruiting energetically advantageous partners with high helix-propensity among the six possible salt-bridge pairs. Third, ME-residues that perform intricate interactions have increased their energy contribution by making major changes in their binary properties. The use of salt-bridge partners as ME-residues, and ME-residues' overlapping usage, predominant in helices, and energetically favorable substitution are some of the favorable features of PfP compared to HuP. These changes in PfP reduce the unfavorable, increase the favorable ME-energy. Thus, the per salt-bridge stability of PfP is greater than that of HuP. Further, unfavorable target ME-residues can be identified whose mutation can increase the stability of salt-bridge. The study applies to other similar systems.

Commercial applications of β-glucosidase (BGL) demands its purity and availability on a large scale. In the present study, we aim to optimize the expression and secretion of a thermostable BGL from Pyrococcus furiosus (PfuBGL) in B. subtilis strain RIK1285. Initial studies with base strain BV002 harboring aprE signal peptide (aprESP) showed PfuBGL yield of 0.743 ± 0.19 pNP U/ml only. A library of 173 different homologous SPs from B. subtilis 168 genome was fused with target PfuBGL gene (PF0073) in pBE-S vector and extracellularly expressed in RIK1285 strain to identify optimal SP for PfuBGL secretion. High-throughput screening of the resulting SP library for BGL activity with a synthetic substrate followed by systematic scaling of the clones yielded a gene construct with CitHSP reporting a sixteen fold enhancement of PfuBGL secretion in comparison to base strain. Batch fermentation (7.5 L scale) PfuBGL yield of the BV003 strain with CitHSP-PF0073 fusion was observed to be 12.08 ± 0.21 pNP U/ml with specific activity of 35.52 ± 0.53 U/mg. Thus, the study represents report on the secretory expression of thermostable PfuBGL using B. subtilis as a host organism and demonstrating its high potential for industrial production of any protein/enzyme., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Infectious diseases caused by viruses such as SARS-CoV-2 and HPV have greatly endangered human health. The nucleic acid detection is essential for the early diagnosis of diseases. Here, we propose a method called PLCR (PfAgo coupled with modified Ligase Chain Reaction for nucleic acid detection) which utilizes PfAgo to only use DNA guides longer than 14-mer to specifically cleave DNA and LCR to precisely distinguish single-base mismatch. PLCR can detect DNA or RNA without PCR at attomolar sensitivities, distinguish single base mutation between the genome of wild type SARS-CoV-2 and its mutant spike D614G, effectively distinguish the novel coronavirus from other coronaviruses and finally achieve multiplexed detection in 70 min. Additionally, LCR products can be directly used as DNA guides without additional input guides to simplify primer design. With desirable sensitivity, specificity and simplicity, the method can be extended for detecting other pathogenic microorganisms., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100°C), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis , a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis -trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90°C for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

Spontaneous deamination of DNA cytosine and adenine into uracil and hypoxanthine, respectively, causes C to T and A to G transition mutations if left unrepaired. Endonuclease Q (EndoQ) initiates the repair of these premutagenic DNA lesions in prokaryotes by cleaving the phosphodiester backbone 5' of either uracil or hypoxanthine bases or an apurinic/apyrimidinic (AP) lesion generated by the excision of these damaged bases. To understand how EndoQ achieves selectivity toward these structurally diverse substrates without cleaving undamaged DNA, we determined the crystal structures of Pyrococcus furiosus EndoQ bound to DNA substrates containing uracil, hypoxanthine, or an AP lesion. The structures show that substrate engagement by EndoQ depends both on a highly distorted conformation of the DNA backbone, in which the target nucleotide is extruded out of the helix, and direct hydrogen bonds with the deaminated bases. A concerted swing motion of the zinc-binding and C-terminal helical domains of EndoQ toward its catalytic domain allows the enzyme to clamp down on a sharply bent DNA substrate, shaping a deep active-site pocket that accommodates the extruded deaminated base. Within this pocket, uracil and hypoxanthine bases interact with distinct sets of amino acid residues, with positioning mediated by an essential magnesium ion. The EndoQ-DNA complex structures reveal a unique mode of damaged DNA recognition and provide mechanistic insights into the initial step of DNA damage repair by the alternative excision repair pathway. Furthermore, we demonstrate that the unique activity of EndoQ is useful for studying DNA deamination and repair in mammalian systems., Competing Interests: Competing interest statement: D.A.H. and R.S.H. are cofounders, shareholders, and consultants of ApoGen Biotechnologies Inc.

It remains undeciphered how thermophilic enzymes display enhanced stability at elevated temperatures. Taking L-asparaginase from P. furiosus (PfA) as an example, we combined scattering shapes deduced from small-angle X-ray scattering (SAXS) data at increased temperatures with symmetry mates from crystallographic structures to find that heating caused end-to-end association. The small contact point of self-binding appeared to be enabled by a terminal short β-strand in N-terminal domain, Leu 179 -Val-Val-Asn 182 (LVVN). Interestingly, deletion of this strand led to a defunct enzyme, whereas suplementation of the peptide LVVN to the defunct enzyme restored structural frameworkwith mesophile-type functionality. Crystal structure of the peptide-bound defunct enzyme showed that one peptide ispresent in the same coordinates as in original enzyme, explaining gain-of lost function. A second peptide was seen bound to the protein at a different location suggesting its possible role in substrate-free molecular-association. Overall, we show that the heating induced self-assembly of native shapes of PfA led to an apparent super-stable assembly.

Modern day aerobic respiration in mitochondria involving complex I converts redox energy into chemical energy and likely evolved from a simple anaerobic system now represented by hydrogen gas-evolving hydrogenase (MBH) where protons are the terminal electron acceptor. Here we present the cryo-EM structure of an early ancestor in the evolution of complex I, the elemental sulfur (S 0 )-reducing reductase MBS. Three highly conserved protein loops linking cytoplasmic and membrane domains enable scalable energy conversion in all three complexes. MBS contains two proton pumps compared to one in MBH and likely conserves twice the energy. The structure also reveals evolutionary adaptations of MBH that enabled S 0 reduction by MBS catalyzed by a site-differentiated iron-sulfur cluster without participation of protons or amino acid residues. This is the simplest mechanism proposed for reduction of inorganic or organic disulfides. It is of fundamental significance in the iron and sulfur-rich volcanic environments of early earth and possibly the origin of life. MBS provides a new perspective on the evolution of modern-day respiratory complexes and of catalysis by biological iron-sulfur clusters.

Arsenate is a notorious toxicant that is known to disrupt multiple biochemical pathways. Many microorganisms have developed mechanisms to detoxify arsenate using the ArsC-type arsenate reductase, and some even use arsenate as a terminal electron acceptor for respiration involving arsenate respiratory reductase (Arr). ArsC-type reductases have been studied extensively, but the phylogenetically unrelated Arr system is less investigated and has not been characterized from Archaea Here, we heterologously expressed the genes encoding Arr from the crenarchaeon Pyrobaculum aerophilum in the euryarchaeon Pyrococcus furiosus , both of which grow optimally near 100°C. Recombinant P. furiosus was grown on molybdenum (Mo)- or tungsten (W)-containing medium, and two types of recombinant Arr enzymes were purified, one containing Mo (Arr-Mo) and one containing W (Arr-W). Purified Arr-Mo had a 140-fold higher specific activity in arsenate [As(V)] reduction than Arr-W, and Arr-Mo also reduced arsenite [As(III)]. The P. furiosus strain expressing Arr-Mo (the Arr strain) was able to use arsenate as a terminal electron acceptor during growth on peptides. In addition, the Arr strain had increased tolerance compared to that of the parent strain to arsenate and also, surprisingly, to arsenite. Compared to the parent, the Arr strain accumulated intracellularly almost an order of magnitude more arsenic when cells were grown in the presence of arsenite. X-ray absorption spectroscopy (XAS) results suggest that the Arr strain of P. furiosus improves its tolerance to arsenite by increasing production of less-toxic arsenate and nontoxic methylated arsenicals compared to that by the parent. IMPORTANCE Arsenate respiratory reductases (Arr) are much less characterized than the detoxifying arsenate reductase system. The heterologous expression and characterization of an Arr from Pyrobaculum aerophilum in Pyrococcus furiosus provides new insights into the function of this enzyme. From in vivo studies, production of Arr not only enabled P. furiosus to use arsenate [As(V)] as a terminal electron acceptor, it also provided the organism with a higher resistance to arsenate and also, surprisingly, to arsenite [As(III)]. In contrast to the tungsten-containing oxidoreductase enzymes natively produced by P. furiosus , recombinant P. aerophilum Arr was much more active with molybdenum than with tungsten. It is also, to our knowledge, the only characterized Arr to be active with both molybdenum and tungsten in the active site., (Copyright © 2020 American Society for Microbiology.)

Caldicellulosiruptor bescii is the most thermophilic cellulolytic organism yet identified (T opt 78 °C). It grows on untreated plant biomass and has an established genetic system thereby making it a promising microbial platform for lignocellulose conversion to bio-products. Here, we investigated the ability of engineered C. bescii to generate alcohols from carboxylic acids. Expression of aldehyde ferredoxin oxidoreductase (aor from Pyrococcus furiosus) and alcohol dehydrogenase (adhA from Thermoanaerobacter sp. X514) enabled C. bescii to generate ethanol from crystalline cellulose and from biomass by reducing the acetate produced by fermentation. Deletion of lactate dehydrogenase in a strain expressing the AOR-Adh pathway increased ethanol production. Engineered strains also converted exogenously supplied organic acids (isobutyrate and n-caproate) to the corresponding alcohol (isobutanol and hexanol) using both crystalline cellulose and switchgrass as sources of reductant for alcohol production. This is the first instance of an acid to alcohol conversion pathway in a cellulolytic microbe.

L-Asparaginase (L-ASNase EC 3.5.1.1) is considered as an important biopharmaceutical drug enzyme in the treatment of childhood acute lymphoblastic leukemia (ALL). In the present study, Pyrococcus furiosus L-ASNase gene was cloned into pET26b (+), expressed in E. coli BL21(DE3) pLysS, and purified to homogeneity using Ni 2+ chelated Fast Flow Sepharose resin with 5.7 purification fold and 23.9% recovery. The purified enzyme exhibited a molecular weight of ~33,660 Da on SDS-PAGE and showed maximal activity at 50 °C and pH 8.0. It retained 98.3% and 60.7% initial activity after 60 min at 37 °C and 50 °C, respectively. The recombinant enzyme showed highest substrate specificity towards L-ASNase substrate, while no detectable specificity was observed for l-glutamine, urea, and acrylamide at 10 mM concentration. The Km and Vmax of the purified recombinant enzyme as calculated using Lineweaver-Burk plot were determined to be 1.623 mM and 105 μmol min -1  mg -1 , respectively. Human leukemia cell line THP-1 treated with recombinant L-ASNase showed significant morphological changes, and the IC 50 of the purified enzyme was found to be 0.8 IU. Moreover, the purified recombinant L-ASNase induced cytotoxic effects on lung adenocarcinoma A549 and colorectal adenocarcinoma Caco-2 cell lines with IC 50 of 1.78 IU and 30 IU, respectively., Competing Interests: Declaration of competing interest I declare that there is no conflict of interest for this article and there is no financial employment, consultancies, honoraria, stock ownership or options, expert testimony, royalties related to this manuscript. Moreover, I declare that this work did not published elsewhere; did not simultaneously submitted for publication elsewhere and all authors agree to the submission., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Escherichia coli cells contain a homolog of presumed 5-keto-4-deoxyuronate isomerase (KduI) from pectin-degrading soil bacteria, but the catalytic activity of the E. coli protein (o-KduI) was never demonstrated., Methods: The known three-dimensional structure of E. coli o-KduI was compared with the available structures of sugar-converting enzymes. Based on the results of this analysis, sugar isomerization activity of recombinant o-KduI was tested against a panel of D-sugars and their derivatives., Results: The three-dimensional structure of o-KduI exhibits a close similarity with Pyrococcus furiosus cupin-type phosphoglucose isomerase. In accordance with this similarity, o-KduI was found to catalyze interconversion of glucose-6-phosphate and fructose-6-phosphate and, less efficiently, conversion of glucuronate to fructuronate. o-KduI was hexameric in crystals but represented a mixture of inactive hexamers and active dimers in solution and contained a tightly bound Zn 2+ ion. Dilution, substrate binding and Zn 2+ removal shifted the hexamer ⇆ dimer equilibrium to the dimers., Conclusions: Our findings identify o-KduI as a novel phosphosugar isomerase in E. coli, whose activity may be regulated by changes in oligomeric structure., General Significance: More than 5700 protein sequences are annotated as KduI, but their enzymatic activity has not been directly demonstrated. E. coli o-KduI is the first characterized member of this group, and its enzymatic activity was found to be different from the predicted activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Isotopologs are powerful tools for investigating biological systems. We report a biosynthetic-cascade synthesis of Trp isotopologs starting from indole, glycine, and formaldehyde using the enzymes l-threonine aldolase and an engineered β-subunit of tryptophan synthase. This modular route to Trp isotopologs is simple and inexpensive, enabling facile access to these compounds.

Background: The secretory production of recombinant proteins in yeast simplifies isolation and purification but also faces possible complications due to the complexity of the secretory pathway. Therefore, correct folding, maturation and intracellular transport of the recombinant proteins are important processing steps with a higher effort needed for complex and large proteins. The aim of this study was to elucidate the secretion potential of Yarrowia lipolytica for low and high molecular weight β-glycosidases in a comparative cultivation approach., Results: A low sized β-glucosidase from Pyrococcus furiosus (CelB; 55 kDa) and a large sized β-galactosidase isolated from the metagenome (M1; 120 kDa) were integrated into the acid extracellular protease locus using the CRISPR-Cas9 system to investigate the size dependent secretion of heterologous proteins in Y. lipolytica PO1f. The recombinant strains were cultivated in the bioreactor for 78 h and the extra- and intracellular enzyme activities were determined. The secretion of CelB resulted in an extracellular volumetric activity of 187.5 µkat oNPGal /L medium , while a volumetric activity of 2.98 µkat oNPGal /L medium was measured during the M1 production. However, when the amount of functional intra- and extracellular enzyme was investigated, the high molecular weight M1 (85%) was secreted more efficiently than CelB (27%). Real-time PCR experiments showed a linear correlation between the transcript level and extracellular activity for CelB, while a disproportional high mRNA level was observed regarding M1. Interestingly, mass spectrometry data revealed the unexpected secretion of two endogenous intracellular glycolytic enzymes, which is reported for the first time for Y. lipolytica., Conclusion: The results of this study provide deeper insights into the secretion potential of Y. lipolytica. A secretion limitation for the low-size CelB was observed, while the large size M1 enzyme was produced in lower amounts but was secreted efficiently. It was shown for the first time that Y. lipolytica is a promising host for the secretion of heterologous high molecular weight proteins (> 100 kDa), although the total secreted amount has to be increased further.

Type III CRISPR-Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3' protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Synthetic DNA-based data storage systems have received significant attention due to the promise of ultrahigh storage density and long-term stability. However, all known platforms suffer from high cost, read-write latency and error-rates that render them noncompetitive with modern storage devices. One means to avoid the above problems is using readily available native DNA. As the sequence content of native DNA is fixed, one can modify the topology instead to encode information. Here, we introduce DNA punch cards, a macromolecular storage mechanism in which data is written in the form of nicks at predetermined positions on the backbone of native double-stranded DNA. The platform accommodates parallel nicking on orthogonal DNA fragments and enzymatic toehold creation that enables single-bit random-access and in-memory computations. We use Pyrococcus furiosus Argonaute to punch files into the PCR products of Escherichia coli genomic DNA and accurately reconstruct the encoded data through high-throughput sequencing and read alignment.

Methionine adenosyltransferases catalyse the biosynthesis of S-adenosylmethionine, the primary methyl group donor in biochemical reactions, through the condensation of methionine and ATP. Here, we report the structural analysis of the Pyrococcus furiosus methionine adenosyltransferase (PfMAT) captured in the unliganded, substrate- and product-bound states. The conformational changes taking place during the enzymatic catalytic cycle are allosterically propagated by amino acid residues conserved in the archaeal orthologues to induce an asymmetric dimer structure. The distinct occupancy of the active sites within a PfMAT dimer is consistent with a half-site reactivity that is mediated by a product-induced negative cooperativity. The structures of intermediate states of PfMAT reported here suggest a distinct molecular mechanism for S-adenosylmethionine synthesis in Archaea, likely consequence of the evolutionary pressure to achieve protein stability under extreme conditions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The tungsten containing Aldehyde:ferredoxin oxidoreductases (AOR) offer interesting opportunities for biocatalytic approaches towards aldehyde oxidation and carboxylic acid reduction. The hyperthermophilic archaeon Pyrococcus furiosus encodes five different AOR family members: glyceraldehyde-3-phosphate oxidoreductase (GAPOR), aldehyde oxidoreductase (AOR), and formaldehyde oxidoreductase (FOR), WOR4 and WOR5. GAPOR functions as a glycolytic enzyme and is highly specific for the substrate glyceraldehyde-3-phosphate (GAP). AOR, FOR and WOR5 have a broad substrate spectrum, and for WOR4 no substrate has been identified to date. As ambiguous kinetic parameters have been reported for different AOR family enzymes the steady state kinetics under different physiologically relevant conditions was explored. The GAPOR substrate GAP was found to degrade at 60 °C by non-enzymatic elimination of the phosphate group to methylglyoxal with a half-life t 1/2  = 6.5 min. Methylglyoxal is not a substrate or inhibitor of GAPOR. D-GAP was identified as the only substrate oxidized by GAPOR, and the kinetics of the enzyme was unaffected by the presence of L-GAP, which makes GAPOR the first enantioselective enzyme of the AOR family. The steady-state kinetics of GAPOR showed partial substrate inhibition, which assumes the GAP inhibited form of the enzyme retains some activity. This inhibition was found to be alleviated completely by a 1 M NaCl resulting in increased enzyme activity at high substrate concentrations. GAPOR activity was strongly pH dependent, with the optimum at pH 9. At pH 9, the substrate is a divalent anion and, therefore, positively charged amino acid residues are likely to be involved in the binding of the substrate. FOR exhibited a significant primary kinetic isotope effect of the apparent Vmax for the deuterated substrate, formaldehyde-d 2 , which shows that the rate-determining step involves a CH bond break from the aldehyde. The implications of these results for the reaction mechanism of tungsten-containing AORs, are discussed., (Copyright © 2019. Published by Elsevier B.V.)

Multimeric enzyme complexes are ubiquitous in nature and catalyze a broad range of useful biological transformations. They are often characterized by a tight allosteric coupling between subunits, making them highly inefficient when isolated. A good example is Tryptophan synthase (TrpS), an allosteric heterodimeric enzyme in the form of an αββα complex that catalyzes the biosynthesis of L-tryptophan. In this study, we decipher the allosteric regulation existing in TrpS from Pyrococcus furiosus ( Pf TrpS), and how the allosteric conformational ensemble is recovered in laboratory-evolved stand-alone β-subunit variants. We find that recovering the conformational ensemble of a subdomain of TrpS affecting the relative stabilities of open, partially closed, and closed conformations is a prerequisite for enhancing the catalytic efficiency of the β-subunit in the absence of its binding partner. The distal mutations resuscitate the allosterically driven conformational regulation and alter the populations and rates of exchange between these multiple conformational states, which are essential for the multistep reaction pathway of the enzyme. Interestingly, these distal mutations can be a priori predicted by careful analysis of the conformational ensemble of the TrpS enzyme through computational methods. Our study provides the enzyme design field with a rational approach for evolving allosteric enzymes toward improved stand-alone function for biosynthetic applications.

The enzymatic microenvironment can impact biocatalytic activity; however, these effects can be difficult to investigate as mutations and fusions can introduce multiple variables and overlapping effects. The fusion of a supercharged protein is a potentially facile means to alter the enzymatic microenvironment. We have investigated complexes made between a thermostable alcohol dehydrogenase (AdhD) and superfolding green fluorescent protein (sfGFP) mutants with extreme surface charges. Three charged sfGFP variants, -30, 0, and +36 were covalently attached to AdhD through the SpyCatcher/SpyTag system. Specific rates for the NAD + -dependent oxidation of butane-2,3-diol were significantly increased in the -30 sfGFP complex, a mixed effect was seen for the 0 sfGFP complexes, and the rates were unaffected by +36 sfGFP complexation. Reactions performed at various pH values (7.8-9.8) and salt concentrations (7.75-500 mm) showed that there was a complex interplay between these effects that was consistent with fusion proteins affecting the local ionic strength, as opposed to the local pH. Steady-state kinetic analyses were performed with the -30 and 0 AdhD-sfGFP complexes. The overall catalytic efficiency was dependent on the charge of the fused sfGFP variant; the -30 sfGFP fusions exhibited the largest beneficial effects at pH 8.8. The impact of the fusions on the apparent ionic strength provides further insight into the effects of charged patches observed on metabolon-forming enzyme complexes., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.

A series of viologen related redox mediators of varying reduction potential has been characterized and their utility as electron shuttles between CdSe quantum dots and hydrogenase enzyme has been demonstrated. Tuning the mediator LUMO energy optimizes the performance of this hybrid photocatalytic system by balancing electron transfer rates of the shuttle.

Soluble hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus is a heterotetrameric [NiFe] hydrogenase that catalyzes the reversible reduction of protons by NADPH into hydrogen gas (H 2 ). Here, the authors expressed the four αβγδ subunits of SHI encoded by one gene cluster in another hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which uses its hydrogenase maturation apparatus without the coexpression of native P. furiosus hydrogenase endopeptidases (maturation proteases). The SHI overexpression of T. kodakarensis resulted in more than 1200-fold enhancement in the hydrogenase activity of the cell lysate compared to that of the host strain with an empty vector. An active, purified 12-His tagged recombinant SHI (rSHI) is obtained by one-step affinity adsorption on nickel-charged resin. Size-exclusion chromatography show that purified rSHI is heterotetrameric and has a molecular mass of 150 kDa. The purified rSHI has a half-life of 70 h at 80 °C. This rSHI is used to design a novel in vitro synthetic enzymatic biosystem to convert pyruvate and H 2 gas into lactate in a theoretical yield, whereas rSHI is used for NADPH regeneration; an FMN-containing diaphorase (DI) is used to match NADP-preferred SHI and NAD-preferred lactate dehydrogenase (LDH). This study provides a cost-efficient method to obtain hyperthermostable hydrogenases, which can be used in in vitro synthetic enzymatic biosystems for cofactor regeneration and hydrogen production., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Enzymes in the prolyl oligopeptidase family possess unique structures and substrate specificities that are important for their biological activity and for potential biocatalytic applications. The crystal structures of Pyrococcus furiosus ( Pfu) prolyl oligopeptidase (POP) and the corresponding S477C mutant were determined to 1.9 and 2.2 Å resolution, respectively. The wild type enzyme crystallized in an open conformation, indicating that this state is readily accessible, and it contained bound chloride ions and a prolylproline ligand. These structures were used as starting points for molecular dynamics simulations of Pfu POP conformational dynamics. The simulations showed that large-scale domain opening and closing occurred spontaneously, providing facile substrate access to the active site. Movement of the loop containing the catalytically essential histidine into a conformation similar to those found in structures with fully formed catalytic triads also occurred. This movement was modulated by chloride binding, providing a rationale for experimentally observed activation of POP peptidase catalysis by chloride. Thus, the structures and simulations reported in this study, combined with existing biochemical data, provide a number of insights into POP catalysis.

Pyrococcus furiosus exoamylase-cum-4-α-glucanotransferase (4-α-GTase; PF0272; PfuAmyGT) is reported to both (i) act upon starch, and (ii) catalyze 'disproportionation' of maltooligosaccharides (with glucose as the smallest product). PfuAmyGT shares ∼65% sequence identity with a homo-dimeric Thermococcus litoralis 4-α-GTase, for which structures are available in complex with a non-hydrolysable analog of maltotetraose (acarbose) bound to one subunit and maltose (of unknown origin) bound to the other subunit. We structurally transposed the maltose onto the acarbose-bound subunit and discovered that the two molecules lie juxtaposed in what could be perfect 'acceptor' and 'donor' substrate-binding sites, respectively. We also discovered that there is a loop between the two sites which could use an available aspartate to excise a glucose from the donor, and an available tryptophan to transfer the glucose to the non-reducing end of the acceptor glucan. We derived a structure for PfuAmyGT through homology-based modeling, identified the potential donor site, acceptor site, glucan-transferring loop, and catalytically important residues, and mutated these to alanine to examine effect(s) upon activity. Mutation D362A abolished creation of shorter, or longer, maltooligosaccharides. Mutation W365A abolished creation of longer oligosaccharides. Mutation H366A had no effect on activity. We propose that D362 facilitates glucose excision, and that W365 facilitates its transfer, either (a) directly into solution (allowing PfuAmyGT to act as an exoamylase), or (b) by glycoside bond formation with an acceptor (allowing PfuAmyGT to act as a 4-α-glucanotransferase), depending upon whether the acceptor site is vacant or occupied in a reaction cycle., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Diverse CRISPR-Cas immune systems protect archaea and bacteria from viruses and other mobile genetic elements. All CRISPR-Cas systems ultimately function by sequence-specific destruction of invading complementary nucleic acids. However, each CRISPR system uses compositionally distinct crRNP [CRISPR (cr) RNA/Cas protein] immune effector complexes to recognize and destroy invasive nucleic acids by unique molecular mechanisms. Previously, we found that Type I-A (Csa) effector crRNPs from Pyrococcus furiosus function in vivo to eliminate invader DNA. Here, we reconstituted functional Type I-A effector crRNPs in vitro with recombinant Csa proteins and synthetic crRNA and characterized properties of crRNP assembly, target DNA recognition and cleavage. Six proteins (Csa 4-1, Cas3″, Cas3', Cas5a, Csa2, Csa5) are essential for selective target DNA binding and cleavage. Native gel shift analysis and UV-induced RNA-protein crosslinking demonstrate that Cas5a and Csa2 directly interact with crRNA 5' tag and guide sequences, respectively. Mutational analysis revealed that Cas3″ is the effector nuclease of the complex. Together, our results indicate that DNA cleavage by Type I-A crRNPs requires crRNA-guided and protospacer adjacent motif-dependent target DNA binding to unwind double-stranded DNA and expose single strands for progressive ATP-dependent 3'-5' cleavage catalyzed by integral Cas3' helicase and Cas3″ nuclease crRNP components.

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions take place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep) significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T 2 generation. Reversed phase high-performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T 2 lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.

The gene-encoding Indole-3-glycerol phosphate synthase, a key enzyme involved in the cyclization of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate, from Pyrococcus furiosus was cloned and expressed in Escherichia coli. The gene product was produced in the soluble and active form. The recombinant protein, purified to apparent homogeneity, displayed highest activity at 100 °C and pH of 5.5. The recombinant enzyme followed Michaelis-Menten kinetics exhibiting apparent V max and K m values of 20 ± 0.5 μmol min -1 mg -1 and 140 ± 10 µM, respectively. The activation energy, determined from the linear Arrhenius plot, was 17 ± 0.5 kJ mol -1 . A unique property of PfInGPS is its stability against denaturants and temperature. There was no significant change in activity even in the presence of 8 M urea or 5 M guanidine hydrochloride. Furthermore, recombinant PfInGPS was highly thermostable with a half-life of 200 min at 100 °C. To the best of our knowledge, this is the most stable indole-3-glycerol phosphate synthase characterized to date.

Glucosamine (GlcN) is widely used in the nutraceutical and pharmaceutical industries. Currently, GlcN is mainly produced by traditional multistep chemical synthesis and acid hydrolysis, which can cause severe environmental pollution, require a long prodution period but a lower yield. The aim of this work was to develop a whole-cell biocatalytic process for the environment-friendly synthesis of glucosamine (GlcN) from N -acetylglucosamine (GlcNAc). We constructed a recombinant Escherichia coli and Bacillus subtilis strains as efficient whole-cell biocatalysts via expression of diacetylchitobiose deacetylase (Dac ph ) from Pyrococcus furiosus . Although both strains were biocatalytically active, the performance of B. subtilis was better. To enhance GlcN production, optimal reaction conditions were found: B. subtilis whole-cell biocatalyst 18.6 g/l, temperature 40°C, pH 7.5, GlcNAc concentration 50 g/l and reaction time 3 h. Under the above conditions, the maximal titer of GlcN was 35.3 g/l, the molar conversion ratio was 86.8% in 3-L bioreactor. This paper shows an efficient biotransformation process for the biotechnological production of GlcN in B. subtilis that is more environmentally friendly than the traditional multistep chemical synthesis approach. The biocatalytic process described here has the advantage of less environmental pollution and thus has great potential for large-scale production of GlcN in an environment-friendly manner.

Noncanonical amino acids (ncAAs) with dual stereocenters at the α and β positions are valuable precursors to natural products and therapeutics. Despite the potential applications of such bioactive β-branched ncAAs, their availability is limited due to the inefficiency of the multistep methods used to prepare them. Herein we report a stereoselective biocatalytic synthesis of β-branched tryptophan analogues using an engineered variant of Pyrococcus furiosus tryptophan synthase (PfTrpB), PfTrpB 7E6 . PfTrpB 7E6 is the first biocatalyst to synthesize bulky β-branched tryptophan analogues in a single step, with demonstrated access to 27 ncAAs. The molecular basis for the efficient catalysis and broad substrate tolerance of PfTrpB 7E6 was explored through X-ray crystallography and UV/Vis spectroscopy, which revealed that a combination of active-site and remote mutations increase the abundance and persistence of a key reactive intermediate. PfTrpB 7E6 provides an operationally simple and environmentally benign platform for the preparation of β-branched tryptophan building blocks., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hyperthermophilic archaea contain a hydrogen gas-evolving,respiratory membrane-bound NiFe-hydrogenase (MBH) that is very closely related to the aerobic respiratory complex I. During growth on elemental sulfur (S°), these microorganisms also produce a homologous membrane-bound complex (MBX), which generates H 2 S. MBX evolutionarily links MBH to complex I, but its catalytic function is unknown. Herein, we show that MBX reduces the sulfane sulfur of polysulfides by using ferredoxin (Fd) as the electron donor, and we rename it membrane-bound sulfane reductase (MBS). Two forms of affinity-tagged MBS were purified from genetically engineered Pyrococcus furiosus (a hyperthermophilic archaea species): the 13-subunit holoenzyme (S-MBS) and a cytoplasmic 4-subunit catalytic subcomplex (C-MBS). S-MBS and C-MBS reduced dimethyl trisulfide (DMTS) with comparable K m (∼490 μm) and V max values (12 μmol/min/mg). The MBS catalytic subunit (MbsL), but not that of complex I (NuoD), retains two of four NiFe-coordinating cysteine residues of MBH. However, these cysteine residues were not involved in MBS catalysis because a mutant P. furiosus strain (MbsLC85A/C385A) grew normally with S°. The products of the DMTS reduction and properties of polysulfides indicated that in the physiological reaction, MBS uses Fd ( E o ' = -480 mV) to reduce sulfane sulfur ( E o ' -260 mV) and cleave organic (RS n R, n ≥ 3) and anionic polysulfides (S n 2- , n ≥ 4) but that it does not produce H 2 S. Based on homology to MBH, MBS also creates an ion gradient for ATP synthesis. This work establishes the electrochemical reaction catalyzed by MBS that is intermediate in the evolution from proton- to quinone-reducing respiratory complexes., (© 2018 Wu et al.)

The 'Bayesian inference of electron microscopy' (BioEM) framework makes it possible to determine the stoichiometry of protein complexes using 3D coarse-grained models and a relatively small number of cryo-electron microscopy images as input. We applied the method to determine the most probable rotor ring stoichiometry of the archaeal Na+ ATP synthase from Pyrococcus furiosus, a multisubunit complex able to produce ATP under extreme conditions. Archaeal ATP synthases consist of a catalytic A1 part and a membrane-embedded AO portion. The AO portion is composed of a rotor ring and the a-subunit. The rotor ring of P. furiosus ATP synthase is composed of 16-kDa c-subunits, each consisting of four helices forming a bundle, with only one Na+-binding site per bundle. This ring was proposed to be decameric from LILBID-MS analysis of the entire ATP synthase. By contrast, the BioEM posterior favors a c9 ring stoichiometry. With BioEM, we ranked coarse-grained models of the whole complex with different ring geometry, using 6400 unprocessed particle images of the A1AO complex collected in vitreous ice. BioEM makes it possible to probabilistically establish the domain stoichiometry using low-resolution information and comparably few particle images.

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN's function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3'-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3'-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.

The NADPH-dependent cytoplasmic [NiFe]-hydrogenase (SHI) from the hyperthermophile Pyrococcus furiosus, which grows optimally near 100°C, is extremely thermostable and has many in vitro applications, including cofactor generation and hydrogen production. In particular, SHI is used in a cell-free synthetic pathway that contains more than a dozen other enzymes and produces three times more hydrogen (12 H2/glucose) from sugars compared to cellular fermentations (4 H2/glucose). We previously reported homologous over-expression and rapid purification of an affinity-tagged (9x-His) version of SHI, which is a heterotetrameric enzyme. However, about 30% of the enzyme that was purified contained an inactive trimeric form of SHI lacking the catalytic [NiFe]-containing subunit. Herein, we constructed a strain of P. furiosus that contained a second set of the eight genes involved in the maturation of the catalytic subunit and insertion of the [NiFe]-site, along with a second set of the four genes encoding the SHI structural subunits. This resulted in a 40% higher yield of the purified affinity-tagged enzyme and the content of the inactive trimeric form decreased to 5% of the total protein. These results bode well for the future production of active SHI for both basic and applied purposes.

Several archaeal species prevalent in extreme environments are particularly exposed to factors likely to cause DNA damages. These include hyperthermophilic archaea (HA), living at temperatures >70°C, which arguably have efficient strategies and robust genome guardians to repair DNA damage threatening their genome integrity. In contrast to Eukarya and other archaea, homologous recombination appears to be a vital pathway in HA, and the Mre11-Rad50 complex exerts a broad influence on the initiation of this DNA damage response process. In a previous study, we identified a physical association between the Proliferating Cell Nuclear Antigen (PCNA) and the Mre11-Rad50 (MR) complex. Here, by performing co-immunoprecipitation and SPR analyses, we identified a short motif in the C- terminal portion of Pyrococcus furiosus Mre11 involved in the interaction with PCNA. Through this work, we revealed a PCNA-interaction motif corresponding to a variation on the PIP motif theme which is conserved among Mre11 sequences of Thermococcale species. Additionally, we demonstrated functional interplay in vitro between P. furiosus PCNA and MR enzymatic functions in the DNA end resection process. At physiological ionic strength, PCNA stimulates MR nuclease activities for DNA end resection and promotes an endonucleolytic incision proximal to the 5' strand of double strand DNA break.