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Hyperthermophilic Sulfolobus solfataricus β-glycosidase (SS-βGly), with higher stability and activity than mesophilic enzymes, has potential for industrial ginsenosides biotransformation. However, its relatively low ginsenoside Rd-hydrolyzing activity limits the production of pharmaceutically active minor ginsenoside compound K (CK). In this study, first, we used molecular docking to predict the key enzyme residues that may hypothetically interact with ginsenoside Rd. Then, based on sequence alignment and alanine scanning mutagenesis approach, key variant sites were identified that might improve the enzyme catalytic efficiency. The enzyme catalytic efficiency (k cat /K m ) and substrate affinity (K m ) of the N264D variant enzyme for ginsenoside Rd increased by 60% and decreased by 17.9% compared with WT enzyme, respectively, which may be due to a decrease in the binding free energy (∆G) between the variant enzyme and substrate Rd. In addition, Markov state models (MSM) analysis during the whole 1000-ns MD simulations indicated that altering N264 to D made the variant enzyme achieve a more stable SS-βGly conformational state than the wild-type (WT) enzyme and corresponding Rd complex. Under identical conditions, the relative activities and the CK conversion rates of the N264D enzyme were 1.7 and 1.9 folds higher than those of the WT enzyme. This study identified an excellent hyperthermophilic β-glycosidase candidate for industrial biotransformation of ginsenosides., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA4 rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA4 phosphodiester hydrolysis in a non-concerted manner., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium-porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The Y-family DNA polymerases specialize in translesion DNA synthesis, which is essential for replicating damaged DNA. The Y-family polymerases, which are made up of four stable domains, exhibit extensive distributions of charged residues, and are responsible for the tight formation of the protein-DNA complex. However, it is still unclear how the electrostatic interactions influence the conformational dynamics of the polymerases. Here, we focus on the case of a prototype Y-family DNA polymerase, Dpo4. Using coarse-grained models including a salt-dependent electrostatic potential, we investigate the effects of the electrostatic interactions on the folding process of Dpo4. Our simulations show that strong electrostatic interactions result in a three-state folding of Dpo4, consistent with the experimental observations. This folding process exhibits low cooperativity led by low salt concentration, where the individual domains fold one by one through one single pathway. Since the refined folding order of domains in multidomain proteins can shrink the configurational space, we suggest that the electrostatic interactions facilitate the Dpo4 folding. In addition, we study the local conformational dynamics of Dpo4 in terms of fluctuation and frustration analyses. We show that the electrostatic interactions can exaggerate the local conformational properties, which are in favor of the large-scale conformational transition of Dpo4 during the functional DNA binding. Our results underline the importance of electrostatic interactions in the conformational dynamics of Dpo4 at both the global and local scale, providing useful guidance in protein engineering at the multidomain level.

Conformational dynamics are important factors in the function of enzymes, including protein tyrosine phosphatases (PTPs). Crystal structures of PTPs first revealed the motion of a protein loop bearing a conserved catalytic aspartic acid, and subsequent nuclear magnetic resonance and computational analyses have shown the presence of motions, involved in catalysis and allostery, within and beyond the active site. The tyrosine phosphatase from the thermophilic and acidophilic Sulfolobus solfataricus (SsoPTP) displays motions of its acid loop together with dynamics of its phosphoryl-binding P-loop and the Q-loop, the first instance of such motions in a PTP. All three loops share the same exchange rate, implying their motions are coupled. Further evidence of conformational flexibility comes from mutagenesis, kinetics, and isotope effect data showing that E40 can function as an alternate general acid to protonate the leaving group when the conserved acid, D69, is mutated to asparagine. SsoPTP is not the first PTP to exhibit an alternate general acid (after VHZ and TkPTP), but E40 does not correspond to the sequence or structural location of the alternate general acids in those precedents. A high-resolution X-ray structure with the transition state analogue vanadate clarifies the role of the active site arginine R102, which varied in structures of substrates bound to a catalytically inactive mutant. The coordinated motions of all three functional loops in SsoPTP, together with the function of an alternate general acid, suggest that catalytically competent conformations are present in solution that have not yet been observed in crystal structures.

Nucleobases within DNA are attacked by reactive oxygen species to produce 7,8-dihydro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as major oxidative lesions. The high mutagenicity of oxoG is attributed to the lesion's ability to adopt syn-oxoG:anti-dA with Watson-Crick-like geometry. Recent studies have revealed that Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) inserts nucleotide opposite oxoA in an error-prone manner and accommodates syn-oxoA:anti-dGTP with Watson-Crick-like geometry, highlighting a promutagenic nature of oxoA. To gain further insights into the bypass of oxoA by Dpo4, we have conducted kinetic and structural studies of Dpo4 extending oxoA:dT and oxoA:dG by incorporating dATP opposite templating dT. The extension past oxoA:dG was ∼5-fold less efficient than that past oxoA:dT. Structural studies revealed that Dpo4 accommodated dT:dATP base pair past anti-oxoA:dT with little structural distortion. In the Dpo4-oxoA:dG extension structure, oxoA was in an anti conformation and did not form hydrogen bonds with the primer terminus base. Unexpectedely, the dG opposite oxoA exited the primer terminus site and resided in an extrahelical site, where it engaged in minor groove contacts to the two immediate upstream bases. The extrahelical dG conformation appears to be induced by the stabilization of anti-oxoA conformation via bifurcated hydrogen bonds with Arg332. This unprecedented structure suggests that Dpo4 may use Arg332 to sense 8-oxopurines at the primer terminus site and slow the extension from the mismatch by promoting anti conformation of 8-oxopurines., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

In the field of biocatalysis and the development of a bio-based economy, hemicellulases have attracted great interest for various applications in industrial processes. However, the study of the catalytic activity of the lignocellulose-degrading enzymes needs to be improved to achieve the efficient hydrolysis of plant biomasses. In this framework, hemicellulases from hyperthermophilic archaea show interesting features as biocatalysts and provide many advantages in industrial applications thanks to their stability in the harsh conditions encountered during the pretreatment process. However, the hemicellulases from archaea are less studied compared to their bacterial counterpart, and the activity of most of them has been barely tested on natural substrates. Here, we investigated the hydrolysis of xyloglucan oligosaccharides from two different plants by using, both synergistically and individually, three glycoside hydrolases from Saccharolobus solfataricus : a GH1 β-gluco-/β-galactosidase, a α-fucosidase belonging to GH29, and a α-xylosidase from GH31. The results showed that the three enzymes were able to release monosaccharides from xyloglucan oligosaccharides after incubation at 65 °C. The concerted actions of β-gluco-/β-galactosidase and the α-xylosidase on both xyloglucan oligosaccharides have been observed, while the α-fucosidase was capable of releasing all α-linked fucose units from xyloglucan from apple pomace, representing the first GH29 enzyme belonging to subfamily A that is active on xyloglucan.

Retaining glycosidase mutants lacking its general acid/base catalytic residue are originally termed thioglycoligases which synthesize thio-linked disaccharides using sugar acceptor bearing a nucleophilic thiol group. A few thioglycoligases derived from retaining α-glycosidases have been classified into a new class of catalysts, O-glycoligases which transfer sugar moiety to a hydroxy group of sugar acceptors, resulting in the formation of O-linked glycosides or oligosaccharides. In this study, an efficient O-α-glucosylation of flavonoids was developed using an O-α-glycoligase derived from a thermostable α-glucosidase from Sulfolobus solfataricus (MalA-D416A). The O-glycoligase exhibited efficient transglycosylation activity with a broad substrate spectrum for all kinds of tested flavonoids including flavone, flavonol, flavanone, flavanonol, flavanol and isoflavone classes in yields of higher than 90%. The glucosylation by MalA-D416A preferred alkaline conditions, suggesting that pH-promoted deprotonation of hydroxyl groups of the flavonoids would accelerate turnover of covalent enzyme intermediate via transglucosylation. More importantly, the glucosylation of flavonoids by MalA-D416A was exclusively regioselective, resulting in the synthesis of flavonoid 7-O-α-glucosides as the sole product. Kinetic analysis and molecular dynamics simulations provided insights into the acceptor specificity and the regiospecificity of O-α-glucosylation by MalA-D416A. This pH promoted transglycosylation using O-α-glycoligases may prove to be a general synthesis route to flavonoid O-α-glycosides., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The β-glucosidase, which hydrolyzes the β(1-4) glucosidic linkage of disaccharides, oligosaccharides and glucose-substituted molecules, has been used in many biotechnological applications. The current commercial source of β-glucosidase is mainly microbial fermentation. Plants have been developed as bioreactors to produce various kinds of proteins including β-glucosidase because of the potential low cost. Sulfolobus solfataricus is a thermoacidophilic archaeon that can grow optimally at high temperature, around 80 °C, and pH 2-4. We overexpressed the β-glucosidase gene from S. solfataricus in transgenic tobacco via Agrobacteria-mediated transformation. Three transgenic tobacco lines with β-glucosidase gene expression driven by the rbcS promoter were obtained, and the recombinant proteins were accumulated in chloroplasts, endoplasmic reticulum and vacuoles up to 1%, 0.6% and 0.3% of total soluble protein, respectively. By stacking the transgenes via crossing distinct transgenic events, the level of β-glucosidase in plants could further increase. The plant-expressed β-glucosidase had optimal activity at 80 °C and pH 5-6. In addition, the plant-expressed β-glucosidase showed high thermostability; on heat pre-treatment at 80 °C for 2 h, approximately 70% residual activity remained. Furthermore, wind-dried leaf tissues of transgenic plants showed good stability in short-term storage at room temperature, with β-glucosidase activity of about 80% still remaining after 1 week of storage as compared with fresh leaf. Furthermore, we demonstrated the possibility of using the archaebacterial β-glucosidase gene as a reporter in plants based on alternative β-galactosidase activity.

The aim of this paper is to make the point on the fortieth years study on the β-glycosidase from Sulfolobus solfataricus. This enzyme represents one of the thermophilic biocatalysts, which is more extensively studied as witnessed by the numerous literature reports available since 1980. Comprehensive biochemical studies highlighted its broad substrate specificity for β-d-galacto-, gluco-, and fuco-sides and also showed its remarkable exo-glucosidase and transglycosidase activities. The enzyme demonstrated to be active and stable over a wide range of temperature and pHs, withstanding to several drastic conditions comprising solvents and detergents. Over the years, a great deal of studies were focused on its homotetrameric tridimensional structure, elucidating several structural features involved in the enzyme stability, such as ion pairs and post-translational modifications. Several β-glycosidase mutants were produced in the years in order to understand its peculiar behavior in extreme conditions and/or to improve its functional properties. The β-glycosidase overproduction was also afforded reporting numerous studies dealing with its production in the mesophilic host Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, and Lactococcus lactis. Relevant applications in food, beverages, bioenergy, pharmaceuticals, and nutraceutical fields of this enzyme, both in free and immobilized forms, highlighted its biotechnological relevance., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

CYP119, a bacterial thermophilic protein from the cytochrome P450 superfamily, has previously been observed in three different conformations with different inhibitors bound using X-ray crystallography. The significance of these states in solution and in the function of the enzyme is not well-known. Double electron-electron resonance (DEER) was used to measure distances and distance distributions between spin-labels for populated conformational states in solution. DEER spectroscopy and molecular dynamics for the ligand-free enzyme suggest that the G helix is in a slightly different conformation than seen previously by crystallography, with the F/G loop in a slightly open conformation. Inhibitor-bound samples showed that this conformation remains as the predominant form, but partial conversion is indicated to a more closed conformation of the F/G loop. However, when the enzyme binds to lauric acid, the proposed substrate, it induces the conversion to a state that is characterized by increased disorder. We propose that similar to recent results with soluble CYP3A4, binding of the inhibitor to CYP119 is accompanied by only small changes in the enzyme structure, but substrate binding results in greater heterogeneity in the structure of the F/G loop region.

The process of homologous recombination is heavily dependent on the RecA family of recombinases for repair of DNA double-strand breaks. These recombinases are responsible for identifying homologies and forming heteroduplex DNA between substrate ssDNA and dsDNA templates, activities that are modified by various accessory factors. In this work we describe the biochemical functions of the SsoRal2 recombinase paralog from the crenarchaeon Sulfolobus solfataricus. We found that the SsoRal2 protein is a DNA-independent ATPase that, unlike the other S. solfataricus paralogs, does not bind either ss- or dsDNA. Instead, SsoRal2 alters the ssDNA binding activity of the SsoRadA recombinase in conjunction with another paralog, SsoRal1. In the presence of SsoRal1, SsoRal2 has a modest effect on strand invasion but effectively abrogates strand exchange activity. Taken together, these results indicate that SsoRal2 assists in nucleoprotein filament modulation and control of strand exchange in S. solfataricus., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus . The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 10 5 M -1 s -1 against paraoxon hydrolysis at 70°C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.

The archaeal exosome is a protein complex with phosphorolytic activity. It is built of a catalytically active hexameric ring containing the archaeal Rrp41 and Rrp42 proteins, and a heteromeric RNA-binding platform. The platform contains a heterotrimer of the archaeal Rrp4 and Csl4 proteins (which harbor S1 and KH or Zn-ribbon RNA binding domains), and comprises additional archaea-specific subunits. The latter are represented by the archaeal DnaG protein, which harbors a novel RNA-binding domain and tightly interacts with the majority of the exosome isoforms, and Nop5, known as a part of an rRNA methylating complex and found to associate with the archaeal exosome at late stationary phase. Although in the cell the archaeal exosome exists in different isoforms with heterotrimeric Rrp4-Csl4-caps, in vitro it is possible to reconstitute complexes with defined, homotrimeric caps and to study the impact of each RNA-binding subunit on exoribonucleolytic degradation and on polynucleotidylation of RNA. Here we describe procedures for reconstitution of isoforms of the Sulfolobus solfataricus exosome and for set-up of RNA degradation and polyadenylation assays.

Protein functions are temperature-dependent, but protein structures are usually solved at a single (often low) temperature because of limitations on the conditions of crystal growth or protein vitrification. Here we demonstrate the feasibility of solving cryo-EM structures of proteins vitrified at high temperatures, solve 12 structures of an archaeal ketol-acid reductoisomerase (KARI) vitrified at 4-70 °C, and show that structures of both the Mg 2+ form (KARI:2Mg 2+ ) and its ternary complex (KARI:2Mg 2+ :NADH:inhibitor) are temperature-dependent in correlation with the temperature dependence of enzyme activity. Furthermore, structural analyses led to dissection of the induced-fit mechanism into ligand-induced and temperature-induced effects and to capture of temperature-resolved intermediates of the temperature-induced conformational change. The results also suggest that it is preferable to solve cryo-EM structures of protein complexes at functional temperatures. These studies should greatly expand the landscapes of protein structure-function relationships and enhance the mechanistic analysis of enzymatic functions.

Nicotinamide adenine dinucleotide phosphate (NAD(P)H)-flavin oxidoreductases (flavin reductases) catalyze the reduction of flavin by NAD(P)H and provide the reduced form of flavin mononucleotide (FMN) to flavin-dependent monooxygenases. Based on bioinformatics analysis, we identified a putative flavin reductase gene, sso2055, in the genome of hyperthermophilic archaeon Sulfolobus solfataricus P2, and further cloned this target sequence into an expression vector. The cloned flavin reductase (EC. 1.5.1.30) was purified to homogeneity and characterized further. The purified enzyme exists as a monomer of 17.8 kDa, free of chromogenic cofactors. Homology modeling revealed this enzyme as a TIM barrel, which is also supported by circular dichroism measurements revealing a beta-sheet rich content. The optimal pH for SSO2055 activity was pH 6.5 in phosphate buffer and the highest activity observed was at 120 °C within the measurable temperature. We showed that this enzyme can use FMN and flavin adenine dinucleotide (FAD) as a substrate to generate their reduced forms. The purified enzyme is predicted to be a potential flavin reductase of flavin-dependent monooxygenases that could be involved in the biodesulfurization process of S. solfataricus P2., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

Dpo4 is a representative model of Y-family DNA polymerase and is therefore one of the most intensively studied DNA polymerase. 6 mA, an epigenetic marker, plays important roles in regulation of various biological processes. However, its effects on DNA replication by Dpo4 is completely unknown. Here, we found that 6 mA and its intermediate Hyp inhibits primer extension by Dpo4, showing an obvious blockage just one nucleotide before 6 mA or Hyp. 6 mA reduces dTTP incorporation efficiency, next-base extension efficiency, binding affinity of DNA to Dpo4, binding affinity of dTTP to Dpo4-DNA complex, the fraction of productive Dpo4 or productive ternary complex, and the burst incorporation rate, explaining the inhibition effects of 6 mA on DNA replication by Dpo4. Hyp is similar to G and dCTP is preferentially incorporated opposite Hyp by Dpo4, resulting in A:T to G:C mutation. Relative to dTTP incorporation opposite unmodified A, Hyp reduces dCTP incorporation efficiency, next-base extension efficiency, the priority in extension beyond correct pair, binding affinity of Dpo4 to DNA, binding of dCTP to Dpo4-DNA complex, and the burst incorporation efficiency, explaining the inhibition effects of Hyp on DNA replication by Dpo4. This work provides insight in the effects of epigenetically modified 6 mA and Hyp on DNA replication by a representative Y-family DNA polymerase Dpo4., (Copyright © 2019 Elsevier Inc. All rights reserved.)

DNA replication requires that the duplex genomic DNA strands be separated; a function that is implemented by ring-shaped hexameric helicases in all Domains. Helicases are composed of two domains, an N- terminal DNA binding domain (NTD) and a C- terminal motor domain (CTD). Replication is controlled by loading of helicases at origins of replication, activation to preferentially encircle one strand, and then translocation to begin separation of the two strands. Using a combination of site-specific DNA footprinting, single-turnover unwinding assays, and unique fluorescence translocation monitoring, we have been able to quantify the binding distribution and the translocation orientation of Saccharolobus (formally Sulfolobus ) solfataricus MCM on DNA. Our results show that both the DNA substrate and the C-terminal winged-helix (WH) domain influence the orientation but that translocation on DNA proceeds N-first., Competing Interests: HP, MT No competing interests declared, (© 2019, Perera and Trakselis.)

Repetitive DNA sequences support the formation of structures that can interrupt replication and repair, leading to breaks and mutagenesis. One particularly stable structure is G-quadruplex (G4) DNA, which is four-stranded and formed from tandemly repetitive guanine bases. When folded within a template, G4 interferes with DNA synthesis. Similar to non-duplex structures, DNA base lesions can also halt an advancing replication fork, but the Y-family polymerases solve this problem by bypassing the damage. In order to better understand how guanine-rich DNA is replicated, we have investigated the activity of the model Y-family polymerase, Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), on guanine-rich templates in vitro. We find that Dpo4 progression on templates containing either a single GC-rich hairpin or a G4 DNA structure is greatly reduced and synthesis stalls at the structure. Human polymerase eta (hPol eta) showed the same pattern of stalling at G4; however, and in contrast to Klenow, hPol eta and Dpo4 partially synthesise into the guanine repeat. Substitution of the nucleotide selectivity residue in Dpo4 with alanine permitted ribonucleotide incorporation on unstructured templates, but this further reduced the ability of Dpo4 to synthesise across from the guanine repeats. The advancement of Dpo4 on G4 templates was highest when the reaction was supplied with only deoxycytidine triphosphate, suggesting that high-fidelity synthesis is favoured over misincorporation. Our results are consistent with a model where the Y-family polymerases pause upon encountering G4 structures but have an ability to negotiate some synthesis through tetrad-associated guanines. This suggests that the Y-family polymerases reduce mutagenesis by catalysing the accurate replication of repetitive DNA sequences, but most likely in concert with additional replication and structure resolution activities., (© The Author(s) 2019. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Circular RNAs (circRNAs) differ structurally from other types of RNAs and are resistant against exoribonucleases. Although they have been detected in all domains of life, it remains unclear how circularization affects or changes functions of these ubiquitous nucleic acid circles. The biogenesis of circRNAs has been mostly described as a backsplicing event, but in archaea, where RNA splicing is a rare phenomenon, a second pathway for circRNA formation was described in the cases of rRNAs processing, tRNA intron excision, and Box C/D RNAs formation. At least in some archaeal species, circRNAs are formed by a ligation step catalyzed by an atypic homodimeric RNA ligase belonging to Rnl3 family. In this review, we describe archaeal circRNA transcriptomes obtained using high throughput sequencing technologies on Sulfolobus solfataricus, Pyrococcus abyssi and Nanoarchaeum equitans cells. We will discuss the distribution of circular RNAs among the different RNA categories and present the Rnl3 ligase family implicated in the circularization activity. Special focus is given for the description of phylogenetic distributions, protein structures, and substrate specificities of archaeal RNA ligases., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Triosephosphate isomerase (TIM) barrel proteins have not only a conserved architecture that supports a myriad of enzymatic functions, but also a conserved folding mechanism that involves on- and off-pathway intermediates. Although experiments have proven to be invaluable in defining the folding free-energy surface, they provide only a limited understanding of the structures of the partially folded states that appear during folding. Coarse-grained simulations employing native centric models are capable of sampling the entire energy landscape of TIM barrels and offer the possibility of a molecular-level understanding of the readout from sequence to structure. We have combined sequence-sensitive native centric simulations with small-angle X-ray scattering and time-resolved Förster resonance energy transfer to monitor the formation of structure in an intermediate in the Sulfolobus solfataricus indole-3-glycerol phosphate synthase TIM barrel that appears within 50 μs and must at least partially unfold to achieve productive folding. Simulations reveal the presence of a major and 2 minor folding channels not detected in experiments. Frustration in folding, i.e., backtracking in native contacts, is observed in the major channel at the initial stage of folding, as well as late in folding in a minor channel before the appearance of the native conformation. Similarities in global and pairwise dimensions of the early intermediate, the formation of structure in the central region that spreads progressively toward each terminus, and a similar rate-limiting step in the closing of the β-barrel underscore the value of combining simulation and experiment to unravel complex folding mechanisms at the molecular level., Competing Interests: The authors declare no conflict of interest.

Promiscuous enzymes can be modified by protein engineering, which enables the catalysis of non-native substrates. γ-lactamase Sspg from Sulfolobus solfataricus is an enzyme with high activity, high stability, and pronounced tolerance of high concentrations of the γ-lactam substrate. These characteristics suggest Sspg as a robust enzymatic catalyst for the preparation of optically pure γ-lactam. This study investigated the modification of this enzyme to expand its application toward resolving chiral esters. γ-Lactamase-esterase conversion was performed by employing a three-step method: initial sequence alignment, followed by substrate screening, and protein engineering based on the obtained substrate-enzyme docking results. This process of fine-tuning of chemical groups on substrates has been termed "substrate screening." Steric hindrance and chemical reactivity of the substrate are major concerns during this step, since both are determining factors for the enzyme-substrate interaction. By employing this three-step method, γ-lactamase Sspg was successfully converted into an esterase with high enantioselectivity towards phenylglycidate substrates (E value > 300). However, since both wild-type Sspg and Sspg mutants did not hydrolyze para-nitrophenyl substrates (pNPs), this esterase activity was termed "atypical esterase activity." The γ-lactamase activity and stability of the Sspg mutants were not severely compromised. The proposed method can be applied to find novel multi-functional enzyme catalysts within existing enzyme pools.

A system is described which permits the efficient synthesis of proteins in vitro at high temperature. It is based on the use of an unfractionated cell lysate (S30) from Sulfolobus solfataricus previously well characterized in our laboratory for translation of pretranscribed mRNAs, and now adapted to perform coupled transcription and translation. The essential element in this expression system is a strong promoter derived from the S. solfataricus 16S/23S rRNA-encoding gene, from which specific mRNAs may be transcribed with high efficiency. The synthesis of two different proteins is reported, including the S. solfataricus DNA-alkylguanine-DNA-alkyl-transferase protein ( Ss OGT), which is shown to be successfully labeled with appropriate fluorescent substrates and visualized in cell extracts. The simplicity of the experimental procedure and specific activity of the proteins offer a number of possibilities for the study of structure-function relationships of proteins.

Programmed cell death 5 (PDCD5) is a vital signaling protein in the apoptosis pathway in eukaryotes. It is known that there are two dissociated N-terminal regions and a triple-helix core in eukaryotic PDCD5. Structural and functional studies of PDCD5 from hyperthermophilic archaea have been limited to date. Here, the PDCD5 homolog Sso0352 (SsoPDCD5) was identified in Sulfolobus solfataricus, the SsoPDCD5 protein was expressed and crystallized, and the phase was identified by single-wavelength anomalous diffraction. The native SsoPDCD5 crystal belonged to space group C2 and diffracted to 1.49 Å resolution. This is the first crystal structure of a PDCD5 homolog to be solved. SsoPDCD5 shares a similar triple-helix bundle with eukaryotic PDCD5 but has a long α-helix in the N-terminus. A structural search and biochemical data suggest that SsoPDCD5 may function as a DNA-binding protein.

Sulfolobus solfataricus is an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group of Sulfolobales , N -acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of the N -glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood in Archaea , and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening of S. solfataricus extracts allowed the detection of a novel α- N -acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified in Archaea Mass spectrometry analysis of the purified activity showed a protein encoded by the sso2901 gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de- N -acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de- N -acetylase instead. The α-GlcNAcase activity observed in S. solfataricus extracts did occur when SSO2901 was used in combination with an α-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream of sso2901 ( sso2900 ) suggest the involvement of these enzymes in the GlcNAc metabolism in S. solfataricus IMPORTANCE In this study, a preliminary screening of cellular extracts of S. solfataricus allowed the identification of an α- N -acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de- N -acetylase, which, in cooperation with the α-glucosidase, catalyzed the hydrolysis of O-α-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de- N -acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism of Archaea ., (Copyright © 2019 American Society for Microbiology.)

(-)-γ-Lactam ((-)-2-azabicyclo[2.2.1]hept-5-en-3-one) has attracted increasing attention as the chiral intermediate of carbocyclic nucleosides most of which serve as pharmaceutical agents such as anti-HIV/HBV drugs abacavir and carbovir. So far, developing in vitro (+)-γ-lactamase-mediated biotransformation has been one of the most efficient approaches for the production of (-)-γ-lactam. In this study, the catalytic activity of the (+)-γ-lactamase from Sulfolobus solfataricus P2 was engineered by semi-rational design. Molecular docking and molecular dynamics simulation were carried out to target the key positions relevant to catalytic activity. Nine amino acid residues were selected for site saturation mutagenesis. To expedite the screening process, a sensitive colorimetric high-throughput screening method was established based on the Rimini test which was originally applied to distinguish primary amines from secondary amines. The screening process resulted in the achievement of several efficient mutants: V203N, V203Q, I336H, I336R, and Y388H. Synergy effects led to four final mutants (V203N/I336R, V203N/Y388H, I336R/Y388H, and V203N/I336R/Y388H) with enhanced enzyme activity after the combination of positive single mutants. The best mutant V203N/Y388H/I336R displayed a 21-fold higher enzyme efficiency (k cat /K M ) compared to the wild-type enzyme. The result demonstrated that the biotransformation using the triple mutant as the catalyst reached > 49% conversion and > 99% enantiomeric excess at 80 °C after 2 h, which made it a good catalyst candidate to produce (-)-γ-lactam. The possible mechanism responsible for the improvement in the catalytic activity was explicated by analyzing the protein-ligand binding modes and interaction between the protein and the ligand.

Tryptophan synthase (TrpS) is a heterotetrameric αββα enzyme that exhibits complex substrate channeling and allosteric mechanisms and is a model system in enzymology. In this work, we characterize proposed early and late evolutionary states of TrpS and show that they have distinct quaternary structures caused by insertions-deletions of sequence segments (indels) in the β-subunit. Remarkably, indole hydrophobic channels that connect α and β active sites have re-emerged in both TrpS types, yet they follow different paths through the β-subunit fold. Also, both TrpS geometries activate the α-subunit through the rearrangement of loops flanking the active site. Our results link evolutionary sequence changes in the enzyme subunits with channeling and allostery in the TrpS enzymes. The findings demonstrate that indels allow protein quaternary architectures to escape "minima" in the evolutionary landscape, thereby overcoming the conservational constraints imposed by existing functional interfaces and being free to morph into new mechanistic enzymes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A growing body of evidence supports a steric exclusion and wrapping model for DNA unwinding in which hexameric helicases interact with the excluded single-stranded DNA (ssDNA) in addition to the encircled strand. Interactions with the excluded ssDNA have been shown to be mediated primarily by electrostatic interactions, but base stacking with surface-exposed tyrosine residues is an alternative hypothesis. Here, we mutated several external tyrosine and positively charged residues from full-length Sulfolobus solfataricus MCM along the proposed path of excluded strand binding and assessed their impact on DNA unwinding. Four of the five tyrosine residues had significant decreases in their level of unwinding, and one, Y519A, located within the α/β-α linker region of the C-terminal domain, had the most severe perturbation attributed to the disruption of hexamerization. The Y519 mutant exhibits an enhanced and stabilized secondary structure that is modulated by temperature, binding DNA with a higher apparent affinity and suggesting a pathway for hexameric assembly. Hydrogen/deuterium exchange coupled to mass spectrometry was used to map deuterium uptake differences between wild-type and Y519A apo structures highlighting global differences in solvent accessible areas consistent with altered quaternary structure. Two of the five electrostatic mutants had significantly reduced levels of DNA unwinding and combined with previous mutations better define the exterior binding path. The importance of the electrostatic excluded strand interaction was confirmed by use of morpholino DNA substrates that showed analogous reduced unwinding rates. These results better define the hexameric assembly and influence of the excluded strand interactions in controlling DNA unwinding by the archaeal MCM complex.

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes, using small CRISPR RNAs that direct effector complexes to degrade invading nucleic acids 1-3 . Type III effector complexes were recently demonstrated to synthesize a novel second messenger, cyclic oligoadenylate, on binding target RNA 4,5 . Cyclic oligoadenylate, in turn, binds to and activates ribonucleases and other factors-via a CRISPR-associated Rossman-fold domain-and thereby induces in the cell an antiviral state that is important for immunity. The mechanism of the 'off-switch' that resets the system is not understood. Here we identify the nuclease that degrades these cyclic oligoadenylate ring molecules. This 'ring nuclease' is itself a protein of the CRISPR-associated Rossman-fold family, and has a metal-independent mechanism that cleaves cyclic tetraadenylate rings to generate linear diadenylate species and switches off the antiviral state. The identification of ring nucleases adds an important insight to the CRISPR system.

Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.

The thermoacidophilic archaea Picrophilus torridus and Sulfolobus solfataricus catabolize glucose via a nonphosphorylative Entner-Doudoroff pathway and a branched Entner-Doudoroff pathway, respectively. Key enzymes for these Entner-Doudoroff pathways are the aldolases, 2-keto-3-deoxygluconate aldolase (KDG-aldolase) and 2-keto-3-deoxy-6-phosphogluconate aldolase [KD(P)G-aldolase]. KDG-aldolase from P. torridus (Pt-KDG-aldolase) is highly specific for the nonphosphorylated substrate, 2-keto-3-deoxygluconate (KDG), whereas KD(P)G-aldolase from S. solfataricus [Ss-KD(P)G-aldolase] is an enzyme that catalyzes the cleavage of both KDG and 2-keto-3-deoxy-6-phosphogluconate (KDPG), with a preference for KDPG. The structural basis for the high specificity of Pt-KDG-aldolase for KDG as compared to the more promiscuous Ss-KD(P)G-aldolase has not been analyzed before. In this work, we report the elucidation of the structure of Ss-KD(P)G-aldolase in complex with KDPG at 2.35 Å and that of KDG-aldolase from P. torridus at 2.50 Å resolution. By superimposition of the active sites of the two enzymes, and subsequent site-directed mutagenesis studies, a network of four amino acids, namely, Arg106, Tyr132, Arg237, and Ser241, was identified in Ss-KD(P)G-aldolase that interact with the negatively charged phosphate group of KDPG, thereby increasing the affinity of the enzyme for KDPG. This KDPG-binding network is absent in Pt-KDG-aldolase, which explains the low catalytic efficiency of KDPG cleavage.

ATPase/Helicases and nucleases play important roles in DNA end-resection, a critical step during homologous recombination repair in all organisms. In hyperthermophilic archaea the exo-endonuclease NurA and the ATPase HerA cooperate with the highly conserved Mre11-Rad50 complex in 3' single-stranded DNA (ssDNA) end processing to coordinate repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA-NurA complex. In this study we demonstrate that the NurA exonuclease activity is inhibited by the Sulfolobus solfataricus RecQ-like Hel112 helicase. Inhibition occurs both in the presence and in the absence of HerA, but is much stronger when NurA is in complex with HerA. In contrast, the endonuclease activity of NurA is not affected by the presence of Hel112. Taken together these results suggest that the functional interaction between NurA/HerA and Hel112 is important for DNA end-resection in archaeal homologous recombination.

The cellular replicative DNA polymerases cannot initiate DNA synthesis without a priming 3' OH. During DNA replication, this is supplied in the context of a short RNA primer molecule synthesized by DNA primase. The primase of archaea and eukaryotes, despite having varying subunit compositions, share sequence and structural homology. Intriguingly, archaeal primase has been demonstrated to possess the ability to synthesize DNA de novo, a property shared with the eukaryotic PrimPol enzymes. The dual RNA and DNA synthetic capabilities of the archaeal DNA primase have led to the proposal that there may be a sequential hand-off between these synthetic modes of primase. In the current work, we dissect the functional interplay between DNA and RNA synthetic modes of primase. In addition, we determine the key determinants that govern primer length definition by the archaeal primase. Our results indicate a primer measuring system that functions akin to a caliper., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The need to find alternative bioremediation solutions for organophosphate degradation pushed the research to develop technologies based on organophosphate degrading enzymes, such as phosphotriesterase. The use of free phosphotriesterase poses limits in terms of enzyme reuse, stability, and process development. The heterogenization of enzyme on a support and their use in bioreactors implemented by membranes seems a suitable strategy, thanks to the ability of membranes to compartmentalize, to govern mass transfer, and to provide a microenvironment with tuned physicochemical and structural properties. Usually, hydrophilic membranes are used since they easily guarantee the presence of water molecules needed for the enzyme catalytic activity. However, hydrophobic materials exhibit a larger shelf life and are preferred for the construction of filters and masks. Therefore, in this work, hydrophobic polyvinylidene fluoride (PVDF) porous membranes were used to develop biocatalytic membrane reactors (BMR). The phosphotriesterase-like lactonase (PLL) enzyme ( SsoPox triple mutant from S. solfataricus) endowed with thermostable phosphotriesterase activity was used as model biocatalyst. The enzyme was covalently bound directly to the PVDF hydrophobic membrane or it was bound to magnetic nanoparticles and then positioned on the hydrophobic membrane surface by means of an external magnetic field. Investigation of kinetic properties of the two BMRs and the influence of immobilized enzyme amount revealed that the performance of the BMR was mostly dependent on the amount of enzyme and its distribution on the immobilization support. Magnetic nanocomposite mediated immobilization showed a much better performance, with an observed specific activity higher than 90% compared to grafting of the enzyme on the membrane. Even though the present work focused on phosphotriesterase, it can be easily translated to other classes of enzymes and related applications.

It is important to understand how the catalytic activity of enzymes is related to their conformational flexibility. We have studied this activity-flexibility correlation using the example of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (ssIGPS), which catalyzes the fifth step in the biosynthesis of tryptophan. ssIGPS is a thermostable representative of enzymes with the frequently encountered and catalytically versatile (βα) 8 -barrel fold. Four variants of ssIGPS with increased catalytic turnover numbers were analyzed by transient kinetics at 25 °C, and wild-type ssIGPS was likewise analyzed both at 25 °C and at 60 °C. Global fitting with a minimal three-step model provided the individual rate constants for substrate binding, chemical transformation, and product release. The results showed that in both cases, namely, the application of activating mutations and temperature increase, the net increase in the catalytic turnover number is afforded by acceleration of the product release rate relative to the chemical transformation steps. Measurements of the solvent viscosity effect at 25 °C versus 60 °C confirmed this change in the rate-determining step with temperature, which is in accordance with a kink in the Arrhenius diagram of ssIGPS at ∼40 °C. When rotational diffusion rates of electron paramagnetic spin-labels attached to active site loop β1α1 are plotted in the form of an Arrhenius diagram, kinks are observed at the same temperature. These findings, together with molecular dynamics simulations, demonstrate that a different degree of loop mobility correlates with different rate-limiting steps in the catalytic mechanism of ssIGPS.

The self-labeling protein tags are robust and versatile tools for studying different molecular aspects of cell biology. In order to be suitable for a wide spectrum of experimental conditions, it is mandatory that these systems are stable after the fluorescent labeling reaction and do not alter the properties of the fusion partner. SsOGT-H 5 is an engineered variant alkylguanine-DNA-alkyl-transferase (OGT) of the hyperthermophilic archaeon Sulfolobus solfataricus, and it represents an alternative solution to the SNAP-tag ® technology under harsh reaction conditions. Here we present the crystal structure of SsOGT-H 5 in complex with the fluorescent probe SNAP-Vista Green ® (SsOGT-H 5 -SVG) that reveals the conformation adopted by the protein upon the trans-alkylation reaction with the substrate, which is observed covalently bound to the catalytic cysteine residue. Moreover, we identify the amino acids that contribute to both the overall protein stability in the post-reaction state and the coordination of the fluorescent moiety stretching-out from the protein active site. We gained new insights in the conformational changes possibly occurring to the OGT proteins upon reaction with modified guanine base bearing bulky adducts; indeed, our structural analysis reveals an unprecedented conformation of the active site loop that is likely to trigger protein destabilization and consequent degradation. Interestingly, the SVG moiety plays a key role in restoring the interaction between the N- and C-terminal domains of the protein that is lost following the new conformation adopted by the active site loop in the SsOGT-H 5 -SVG structure. Molecular dynamics simulations provide further information into the dynamics of SsOGT-H 5 -SVG structure, highlighting the role of the fluorescent ligand in keeping the protein stable after the trans-alkylation reaction., (Copyright © 2018 Elsevier Inc. All rights reserved.)

DNA-protein cross-links (DPCs) are important DNA lesions induced by endogenous crosslinking agents such as formaldehyde or acetaldehyde, as well as ionizing radiation, cancer chemotherapeutic drugs, and abortive action of some enzymes. Due to their very bulky nature, they are expected to interfere with DNA and RNA synthesis and DNA repair. DPCs are highly genotoxic and the ability of cells to deal with them is relevant for many chemotherapeutic interventions. However, interactions of DNA polymerases with DPCs have been poorly studied due to the lack of a convenient experimental model. We have used NaBH4-induced trapping of E. coli formamidopyrimidine-DNA glycosylase with DNA to construct model DNA polymerase substrates containing a DPC in single-stranded template, or in the template strand of double-stranded DNA, or in the non-template (displaced) strand of double-stranded DNA. Nine DNA polymerases belonging to families A, B, X, and Y were studied with respect to their behavior upon encountering a DPC: Klenow fragment of E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, Pyrococcus furiosus DNA polymerase, Sulfolobus solfataricus DNA polymerase IV, human DNA polymerases β, κ and λ, and DNA polymerases from bacteriophages T4 and RB69. Although none were able to fully bypass DPCs in any context, Family B DNA polymerases (T4, RB69) and Family Y DNA polymerase IV were able to elongate the primer up to the site of the cross-link if a DPC was located in single-stranded template or in the displaced strand. In other cases, DNA synthesis stopped 4-5 nucleotides before the site of the cross-link in single-stranded template or in double-stranded DNA if the polymerases could displace the downstream strand. We suggest that termination of DNA polymerases on a DPC is mostly due to the unrelieved conformational strain experienced by the enzyme when pressing against the cross-linked protein molecule., Competing Interests: The authors have declared that no competing interests exist.

Background: Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus. To follow this aim new induction approaches were investigated to boost the enzyme production, high cell density fermentation strategies were set-up to reach higher and higher enzyme yields up to 22-L scale, a downstream train was studied to meet the requirements of an efficient industrial purification process., Results: Physiological studies in shake flasks demonstrated that the use of galactose as inducer increased the enzyme concentrations up to 4.5 folds, compared to the production obtained by induction with IPTG. Optimising high cell density fed-batch strategies the production and the productivity of both enzymes were further enhanced of 26 folds, up to 2300 U·L - 1 and 47.1 U·L - 1 ·h - 1 for SacPox and to 8700 U·L - 1 and 180.6 U·L - 1 ·h - 1 for SsoPox C258L/I261F/W263A, and the fermentation processes resulted scalable from 2.5 to 22.0 L. After being produced and extracted from the cells, the enzymes were first purified by a thermo-precipitation step, whose conditions were optimised by response surface methodology. A following ultra-filtration process on 100 and 5 KDa cut-off membranes drove to a final pureness and a total recovery of both enzymes of 70.0 ± 2.0%, suitable for industrial applications., Conclusions: In this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes SacPox and SsoPox C258L/I261F/W263A was set-up. The enzyme production was boosted by combining a new galactose induction approach with high cell density fed-batch fermentation strategies. An efficient enzyme purification protocol was designed coupling a thermo-precipitation step with a following membrane-based ultra-filtration process.

DING proteins represent a new group of 40 kDa-related members, ubiquitous in living organisms. The family also include the DING protein from Sulfolobus solfataricus, functionally related to poly(ADP-ribose) polymerases. Here, the archaeal protein has been compared with the human Phosphate-Binding Protein and the Pseudomonas fluorescence DING enzyme, by enzyme assays and immune cross-reactivity. Surprisingly, as the Sulfolobus enzyme, the Human and Pseudomonas proteins display poly(ADP-ribose) polymerase activity, whereas a phosphatase activity was only present in Sulfolobus and human protein, despite the conserved phosphate-binding site residues in Pseudomonas DING. All proteins were positive to anti-DING antibodies and gave a comparable pattern of anti-poly(ADP-ribose) polymerase immunoreactivity with two bands, at around 40 kDa and roughly at the double of this molecular mass. The latter signal was present in all Sulfolobus enzyme preparations and proved not due to either a contaminant or a precursor protein, but likely being a dimeric form of the 40 kDa polypeptide. The common immunological and partly enzymatic behavior linking human, Pseudomonas and Sulfolobus DING proteins, makes the archaeal protein an important model system to investigate DING protein function and evolution within the cell.

Coproheme decarboxylases (ChdCs) are enzymes responsible for the catalysis of the terminal step in the coproporphyrin-dependent heme biosynthesis pathway. Phylogenetic analyses confirm that the gene encoding for ChdCs is widespread throughout the bacterial world. It is found in monoderm bacteria (Firmicutes, Actinobacteria), diderm bacteria (e. g. Nitrospirae) and also in Archaea. In order to test phylogenetic prediction ChdC representatives from all clades were expressed and examined for their coproheme decarboxylase activity. Based on available biochemical data and phylogenetic analyses a sequence motif (-Y-P-M/F-X-K/R-) is defined for ChdCs. We show for the first time that in diderm bacteria an active coproheme decarboxylase is present and that the archaeal ChdC homolog from Sulfolobus solfataricus is inactive and its physiological role remains elusive. This shows the limitation of phylogenetic prediction of an enzymatic activity, since the identified sequence motif is equally conserved across all previously defined clades., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was investigated in different organic cosolvents by means of kinetic and calorimetric measurements, molecular dynamics simulations, and small-angle X-ray scattering. The combined experimental and theoretical techniques were essential to complement each other's limitations in the investigation of the complex interaction pattern between the enzyme, different solvent types, and concentrations. In this way, the underlying mechanisms for the loss of enzyme activity in different water-miscible solvents could be elucidated. These include direct inhibitory effects onto the active center and structural distortions., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. β-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. β-Glycosidase from S. solfataricus can hydrolyze β-d-glucopyranoside in ginsenosides Rc, C-Mc1, and C-Mc, but not α-l-arabinofuranoside in these ginsenosides. To determine candidate residues involved in α-l-arabinofuranosidase activity, compound Mc (C-Mc) was docking to β-glycosidase from S. solfataricus in homology model and sequence was aligned with β-glycosidase from Pyrococcus furiosus that has α-l-arabinofuranosidase activity. A L213A variant β-glycosidase with increased α-l-arabinofuranosidase activity was selected by substitution of other amino acids for candidate residues. The increased α-l-arabinofuranosidase activity of the L213A variant was confirmed through the determination of substrate specificity, change in binding energy, transformation pathway, and C-K production from ginsenosides Rc and C-Mc. The L213A variant β-glycosidase catalyzed the conversion of Rc to Rd by hydrolyzing α-l-arabinofuranoside linked to Rc, whereas the wild-type β-glycosidase did not. The variant enzyme converted ginsenosides Rc and C-Mc into C-K with molar conversions of 97%, which were 1.5- and 2-fold higher, respectively, than those of the wild-type enzyme. Therefore, protein engineering is a useful tool for enhancing the hydrolytic activity on specific glycoside linked to ginsenosides.

Translesion DNA synthesis is an essential process that helps resume DNA replication at forks stalled near bulky adducts on the DNA. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) that can be metabolically activated to benzo[a]pyrene diol epoxide (BPDE), which then can react with DNA to form carcinogenic DNA adducts. Here, we have used single-molecule florescence resonance energy transfer (smFRET) experiments, classical molecular dynamics simulations, and nucleotide incorporation assays to investigate the mechanism by which the model Y-family polymerase, Dpo4, bypasses a (+)-cis-B[a]P-N 2 -dG adduct in DNA. Our data show that when (+)-cis-B[a]P-N 2 -dG is the templating base, the B[a]P moiety is in a non-solvent exposed conformation stacked within the DNA helix, where it effectively blocks nucleotide incorporation across the adduct by Dpo4. However, when the media contains a small amount of dimethyl sulfoxide (DMSO), the adduct is able to move to a solvent-exposed conformation, which enables error-prone DNA replication past the adduct. When the primer terminates across from the adduct position, the addition of DMSO leads to the formation of an insertion complex capable of accurate nucleotide incorporation.

O ⁶-DNA-alkyl-guanine-DNA-alkyl-transferases (OGTs) are evolutionarily conserved, unique proteins that repair alkylation lesions in DNA in a single step reaction. Alkylating agents are environmental pollutants as well as by-products of cellular reactions, but are also very effective chemotherapeutic drugs. OGTs are major players in counteracting the effects of such agents, thus their action in turn affects genome integrity, survival of organisms under challenging conditions and response to chemotherapy. Numerous studies on OGTs from eukaryotes, bacteria and archaea have been reported, highlighting amazing features that make OGTs unique proteins in their reaction mechanism as well as post-reaction fate. This review reports recent functional and structural data on two prokaryotic OGTs, from the pathogenic bacterium Mycobacterium tuberculosis and the hyperthermophilic archaeon Sulfolobus solfataricus , respectively. These studies provided insight in the role of OGTs in the biology of these microorganisms, but also important hints useful to understand the general properties of this class of proteins., Competing Interests: The authors declare no conflict of interest.

The redesign of enzyme active sites to alter their function or specificity is a difficult yet appealing challenge. Here we used a structure-based design approach to engineer the lactonase SsoPox from Sulfolobus solfataricus into a phosphotriesterase. The five best variants were characterized and their structure was solved. The most active variant, αsD6 (V27A-Y97W-L228M-W263M) demonstrates a large increase in catalytic efficiencies over the wild-type enzyme, with increases of 2,210-fold, 163-fold, 58-fold, 16-fold against methyl-parathion, malathion, ethyl-paraoxon, and methyl-paraoxon, respectively. Interestingly, the best mutants are also capable of degrading fensulfothion, which is reported to be an inhibitor for the wild-type enzyme, as well as others that are not substrates of the starting template or previously reported W263 mutants. The broad specificity of these engineered variants makes them promising candidates for the bioremediation of organophosphorus compounds. Analysis of their structures reveals that the increase in activity mainly occurs through the destabilization of the active site loop involved in substrate binding, and it has been observed that the level of disorder correlates with the width of the enzyme specificity spectrum. This finding supports the idea that active site conformational flexibility is essential to the acquisition of broader substrate specificity.

Although translesion synthesis (TLS) polymerases play key roles in replicating DNA that contains nucleobase addition products (adducts), there are many unknowns about their function. The present work gains indispensable structural insights from molecular dynamics simulations on the replication of O6-benzyl-guanine (Bz-dG) prior to bond formation during dCTP insertion opposite the adduct by Dpo4. When combined with previous X-ray crystal structures of the Bz-dG extension complex, molecular details are now available for each stage during a single TLS replication cycle for this carcinogenic lesion. Our calculations illustrate that Bz-dG preferentially adopts an intercalated bulky moiety orientation in the Dpo4 preinsertion complex, which stabilizes the complex through Bz-dG interactions with the previously replicated 3'-base pair and positions the carcinogenic group in the dNTP binding site. Nevertheless, the maintained inherent flexibility of Bz-dG due to a stark lack of interactions with the polymerase or template DNA allows the bulky moiety to adopt a major groove position opposite an incoming dCTP in an orientation that is conducive for the experimentally observed nonmutagenic bypass. Comparison of Bz-dG and canonical dG replication clarifies that the experimentally observed decrease in dCTP binding affinity and replication efficiency upon adduct formation is likely caused by a combination of factors, including the required template nucleotide conformational change and destabilized template-dCTP hydrogen bonding. Although additional aspects of the replication process, such as the impact of the adduct on the nucleotidyl-transfer reaction, may also be important for fully rationalizing experimental replication data and must be considered in future work, the present contribution emphasizes the importance of considering the effect of DNA damage on the early stages of the TLS process.

The HerA-NurA helicase-nuclease complex cooperates with Mre11 and Rad50 to coordinate the repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA-NurA. By combining hybrid mass spectrometry with cryo-EM, computational and biochemical data, we investigate the oligomeric formation of HerA and detail the mechanism of nucleotide binding to the HerA-NurA complex from thermophilic archaea. We reveal that ATP-free HerA and HerA-DNA complexes predominantly exist in solution as a heptamer and act as a DNA loading intermediate. The binding of either NurA or ATP stabilizes the hexameric HerA, indicating that HerA-NurA is activated by substrates and complex assembly. To examine the role of ATP in DNA translocation and processing, we investigated how nucleotides interact with the HerA-NurA. We show that while the hexameric HerA binds six nucleotides in an 'all-or-none' fashion, HerA-NurA harbors a highly coordinated pairwise binding mechanism and enables the translocation and processing of double-stranded DNA. Using molecular dynamics simulations, we reveal novel inter-residue interactions between the external ATP and the internal DNA binding sites. Overall, here we propose a stepwise assembly mechanism detailing the synergistic activation of HerA-NurA by ATP, which allows efficient processing of double-stranded DNA., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Organophosphorus insecticides (OPs) are toxic compounds used for agricultural purposes and responsible for severe types of contamination worldwide. OPs may also induce chronic deleterious effects and developmental disruption. Finding remediation strategies is a major concern to diminish their impact on environment and human health. Enzymes have emerged as a promising eco-friendly route for decontaminating OPs. The enzyme SsoPox from the archaea Sulfolobus solfataricus has been particularly studied, considering both its tremendous stability and phosphotriesterase activity. However, the toxicity of the degradation products generated through enzyme hydrolysis has been poorly investigated. To address both neurotoxicity and developmental perturbation, freshwater planarians from Platyhelminthes were considered to evaluate the impact of OP and degradation product exposure. Planarians have a large proportion of stem cells that give them an unconventional capacity for regeneration. OPs were found to be highly toxic to planarians and enzyme decontamination drastically enhanced survival rate. Although not completely innocuous, the degradation products were found to be less toxic than insecticides and reduced poisoning effects by increasing NOEC values by up to eight-fold. SsoPox also limited detrimental consequences on planarian mobility and enabled them to recover a non-exposed type regeneration process suggesting that enzymatic decontamination is a promising alternative to bioremediation.

In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate at 30 °C and at 70 °C. At 30 °C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 °C, 1,3-bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. When phosphoglycerate kinase was incubated together with glyceraldehyde 3-phosphate dehydrogenase it was possible to convert 3-phosphoglycerate to glyceraldehyde 3-phosphate, both at 30 °C and at 70 °C, however, at 70 °C only low concentrations of product were observed due to thermal instability of glyceraldehyde 3-phosphate. Thus, thermolabile intermediates challenge central metabolic reactions and require special adaptation strategies for life at high temperature.