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Adenosine triphosphate (ATP) is the energy currency of all living organisms and can be used as an indicator for cell proliferation and cytotoxicity. In the present work, we have developed a novel ATP detection system by combining the biotinylation reaction from archaeon Sulfolobus tokodaii with fluorescence resonance energy transfer (FRET). In biotinylation from S. tokodaii, an enzyme known as biotin protein ligase (BPL) forms a very stable complex with its product, biotinylated substrate protein (BCCP). Here, BPL and BCCP were fused to the fluorescent proteins Cerulean and Clover, respectively, and ATP detection was accomplished by monitoring the FRET signal between the two fluorescent proteins, since ATP is an essential component for biotinylation and the tight BPL-BCCP complex is formed only after biotinylation. Using this system, we have succeeded in detecting 5 nM of ATP by biotinylation reaction with 50 nM of each fusion protein. Our method has a characteristic that the signal does not decay for at least 2 h after the start of the reaction, unlike in the case of the luminescence-based assay with luciferase commonly used for the ATP detection. Thus, our system allows for ATP detection which is not significantly constrained by measurement timing., 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 Inc. All rights reserved.)

Inorganic pyrophosphatase catalyzes the conversion of pyrophosphate to phosphate and is often critical for driving reactions forward in cellular processes such as nucleic acid and protein synthesis. Commonly used methods for quantifying pyrophosphatase enzyme activity employ reacting liberated phosphate with a second molecule to produce absorbance changes or employing a second enzyme in coupled reactions to produce a product with a detectable absorbance. In this investigation, a novel [ 31 P]-NMR spectroscopy-based assay was used to quantitatively measure the formation of phosphate and evaluate the activity of inorganic pyrophosphatase from the thermoacidophilic Crenarchaeota Sulfolobus islandicus. The enzymatic activity was directly measured via integration of the [ 31 P] resonance associated with the phosphate product (δ = 2.1 ppm). Sulfolobus islandicus inorganic pyrophosphatase preferentially utilized Mg 2+ as divalent cation and had pH and temperature optimums of 6.0 of 50 °C, respectively. The V max value was 850 μmol/min/mg and the K m for pyrophosphate was 1.02 mM. Sequence analysis indicates the enzyme is a Family I pyrophosphatase. Sulfolobus islandicus inorganic pyrophosphatase was shown to be inhibited by sodium fluoride with a IC 50 of 2.26 mM, compared to a IC 50 of 0.066 mM for yeast inorganic pyrophosphatase. These studies reveal that a [ 31 P]-NMR spectroscopy-based assay is an effective method for analyzing catalysis by phosphate-producing enzymes., 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 © 2021 Elsevier Inc. All rights reserved.)

Endonuclease III (EndoIII), which is ubiquitous in bacteria, Archaea and eukaryotes, plays an important role in excising thymine glycol (Tg) from DNA. Herein, we present evidence that an EndoIII from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A (Sis-EndoIII) is capable of removing Tg from DNA at high temperature. Biochemical data show that the optimal temperature and pH of Sis-EndoIII are ca.70 °C and ca.7.0-8.0, respectively. Furthermore, the recombinant Sis-EndoIII retains relative weak activity without a divalent metal ion, and displays maximum activity in the presence of Mg 2+ or Ca 2+ . Additionally, we first revealed the activation energy (E a ) of 39.7 ± 4.2 kcal/mol for Sis-EndoIII to remove Tg from dsDNA. As a bifunctional glycosylase, Sis-EndoIII possesses AP lyase activity in addition to glycosylase activity. Additionally, a covalent intermediate is formed between Sis-EndoIII and Tg-containing dsDNA. Mutational studies demonstrate that residues D50, K133 and D151 in Sis-EndoIII are responsible for removal of Tg from dsDNA and K133 and D151 are essential for formation of the covalent intermediate. To our knowledge, it is the first report of Tg excision by crenarchaeal EndoIII, thus augmenting our understanding on archaeal EndoIII function., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Type III CRISPR-cas systems initiate cyclic oligo-adenylate (cOA) signaling to initiate immune response. Previously, we identified that a membrane-associated DHH-DHHA1 family protein from Sulfolobus islandicus efficiently degrades cOA. Here, we provide detailed protocols for expression and purification of the protein from its native host and a cOA degradation assay with the purified enzyme. The methodology should be of interest for researchers studying Sulfolobus , membrane-associated proteins, or type III CRISPR-cas systems. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

In a directed evolution aimed at improving enzymatic activity, a situation occurs where highly active variants can no longer be obtained from a template protein because the template is already located at a peak (local maximum) in the fitness landscape of activity for the sequence space. To overcome this situation, the template needs to descend the mountain (lose activity) once and climb another higher mountain. However, there is no solid guideline of how the template should go down. Here, we propose a stability index. Previous studies have shown that protein evolution is potentially governed by stability, and that proteins with low activity but high stability are more favorable templates for producing highly active variants. In our earlier works on conventional directed evolution by random mutagenesis of an esterase from Sulfolobus tokodaii, we identified variants with 3-fold higher activity than the wild-type as the highest activity variants. In this work, as a first step, stability-keeping variants were selected by five rounds of random mutagenesis and screening based on halo formation assay using the substrate tributyrin at 70 °C after heat treatment for 30 min at 90 °C. These variants are likely to be scattered at the feet of various mountains in the fitness landscape. Next, these variants were pooled and used as parental proteins for a conventional experiment with activity-based selection, where the activity of variants was assayed using their cell-free extracts on the substrate p-nitrophenyl butyrate at 75 °C. After two rounds of random mutagenesis, we successfully obtained a variant with 9-fold higher activity than the wild-type. These results indicate that the two-step selection by stability and activity enables us more easily to produce markedly activity-improving variants., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Type III CRISPR-Cas systems initiate an intracellular signaling pathway to confer immunity. The signaling pathway includes synthesis of cyclic oligo-adenylate (cOA) and activation of the RNase activity of type III accessory ribonuclease Csm6/Csx1 by cOA. After the immune response, cOA should be cleared on time to avoid constant cellular RNA degradation. In this study, we find a metal-dependent cOA degradation activity in Sulfolobus islandicus. The activity is associated with the cell membrane and able to accelerate cOA clearance at a high cOA level. Further, we show that a metal-dependent and membrane-associated DHH-DHHA1 family nuclease (MAD) rapidly cleaves cOA and deactivates Csx1 ribonuclease. The cOA degradation efficiency of MAD is much higher than the cellular ring nuclease. However, the subcellular organization may prevent it from degrading nascent cOA. Together, the data suggest that MAD acts as the second cOA degrader after the ring nuclease to remove diffused redundant cOA., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

A gene encoding a dye-linked d-amino acid dehydrogenase (Dye-DADH) homologue was found in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. The predicted amino acid sequence suggested that the gene product is a membrane-bound type enzyme. The gene was overexpressed in Escherichia coli, but the recombinant protein was exclusively produced as an inclusion body. In order to avoid production of the inclusion body, an expression system using the thermoacidophilic archaeon Sulfolobus acidocaldarius instead of E. coli as the host cell was constructed. The gene was successfully expressed in Sulfolobus acidocaldarius, and its product was purified to homogeneity and characterized. The purified enzyme catalyzed the dehydrogenation of various d-amino acids, with d-phenylalanine being the most preferred substrate. The enzyme retained its full activity after incubation at 90 °C for 30 min and after incubation at pH 4.0-11.0 for 30 min at 50 °C. This is the first report on membrane-bound Dye-DADH from thermophilic archaea that was successfully expressed in an archaeal host., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Aminoacyl-tRNA synthetases (aaRSs) are ancient enzymes that play a fundamental role in protein synthesis. They catalyze the esterification of specific amino acids to the 3'-end of their cognate tRNAs and therefore play a pivotal role in protein synthesis. Although previous studies suggest that aaRS-dependent errors in protein synthesis can be beneficial to some microbial species, evidence that reduced aaRS fidelity can be adaptive is limited. Using bioinformatics analyses, we identified two distinct leucyl-tRNA synthetase (LeuRS) genes within all genomes of the archaeal family Sulfolobaceae. Remarkably, one copy, designated LeuRS-I, had key amino acid substitutions within its editing domain that would be expected to disrupt hydrolytic editing of mischarged tRNA Leu and to result in variation within the proteome of these extremophiles. We found that another copy, LeuRS-F, contains canonical active sites for aminoacylation and editing. Biochemical and genetic analyses of the paralogs within Sulfolobus islandicus supported the hypothesis that LeuRS-F, but not LeuRS-I, functions as an essential tRNA synthetase that accurately charges leucine to tRNA Leu for protein translation. Although LeuRS-I was not essential, its expression clearly supported optimal S. islandicus growth. We conclude that LeuRS-I may have evolved to confer a selective advantage under the extreme and fluctuating environmental conditions characteristic of the volcanic hot springs in which these archaeal extremophiles reside., (© 2020 Weitzel et al.)

Enzyme-catalyzed hydrolysis of echothiophate, a P-S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of Brevundimonas diminuta phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of Brevundimonas diminuta and Sulfolobus solfataricus phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with K m = 0.20 ± 0.03 mM and k cat = 5.4 ± 1.6 min -1 . The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency ( K m = 2.6 ± 0.2 mM; k cat = 53400 min -1 ). With a k cat /K m = (2.6 ± 1.6) × 10 7 M -1 min -1 , GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P-S bonded OPs by thiol-free OP hydrolases.

Two variants of the enzyme family pyruvate:ferredoxin oxidoreductase (PFOR), derived from the anaerobic sulfate-reducing bacterium Desulfovibrio africanus and the extremophilic crenarchaeon Sulfolobus acidocaldarius, respectively, were evaluated for their capacity to fixate CO 2 in vitro. PFOR reversibly catalyzes the conversion of acetyl-CoA and CO 2 to pyruvate using ferredoxin as redox partner. The oxidative decarboxylation of pyruvate is thermodynamically strongly favored, and most previous studies only considered the oxidative direction of the enzyme. To assay the pyruvate synthase function of PFOR during reductive carboxylation of acetyl-CoA is more challenging and requires to maintain the reaction far from equilibrium. For this purpose, a biochemical assay was established where low-potential electrons were introduced by photochemical reduction of EDTA/deazaflavin and the generated pyruvate was trapped by chemical derivatization with semicarbazide. The product of CO 2 fixation could be detected as pyruvate semicarbazone by HPLC-MS. In a combinatorial approach, both PFORs were tested with ferredoxins from different sources. The pyruvate semicarbazone product could be detected with low-potential ferredoxins of the green sulfur bacterium Chlorobium tepidum and of S. acidocaldarius whereas CO 2 fixation was not supported by the native ferredoxin of D. africanus. Methylviologen as an artificial electron carrier also allowed CO 2 fixation. For both enzymes, the results are the first demonstration of CO 2 fixation in vitro. Both enzymes exhibited high stability in the presence of oxygen during purification and storage. In conclusion, the employed PFOR enzymes in combination with non-native ferredoxin cofactors might be promising candidates for further incorporation in biocatalytic CO 2 conversion. ENZYMES: EC1.2.7.1. Pyruvate:Ferredoxin Oxidoreductase., (© 2019 Federation of European Biochemical Societies.)

In our previous study, we investigated the relationship between protein evolution and stability through the random mutational drift of an esterase from hyperthermophilic archaeon Sulfolobus tokodaii. The results revealed that evolvability, which is the appearance frequency of variants with higher activity than the parent protein, correlates with parental stability. This suggests that protein evolution that does not take stability into account does not make sense. Here, we used those data to further evaluate the relationship between activity and stability in random mutations, revealing that the maximum increase in activity due to mutation conflicts with parental stability. That is, many activated variants are produced when parental stability is high, whereas lower stability offers a few excellent variants with much higher activity. Moreover, we used the random mutant library to compute a novel criterion, robustizability (stabilizability), which is the appearance frequency of variants with a higher stability than the parent protein. Robustizability correlates positively with parental activity and negatively with parental stability. The results indicated that the principle of activity-stability trade-off dominates, in even random mutations. We propose its application in protein engineering via directed evolution by stability selection., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The thermo-acidophilic archaeon, Sulfolobus tokodaii, was utilized for the production of Pd(0) bionanoparticles from acidic Pd(II) solution. Use of active cells was essential to form well-dispersed Pd(0) nanoparticles located on the cell surface. The particle size could be manipulated by modifying the concentration of formate (as electron donor; e-donor) and by addition of enzymatic inhibitor (Cu 2+ ) in the range of 14-63 nm mean size. Since robust Pd(II) reduction progressed in pre-grown S. tokodaii cells even in the presence of up to 500 mM Cl - , it was possible to conversely utilize the effect of Cl - to produce even finer and denser particles in the range of 8.7-15 nm mean size. This effect likely resulted from the increasing stability of anionic Pd(II)-chloride complex at elevated Cl - concentrations, eventually allowing involvement of greater number of initial Pd(0) crystal nucleation sites (enzymatic sites). The catalytic activity [evaluated based on Cr(VI) reduction reaction] of Pd(0) bionanoparticles of varying particle size formed under different conditions were compared. The finest Pd(0) bionanoparticles obtained at 50 mM Cl - (mean 8.7 nm; median 5.6 nm) exhibited the greatest specific Cr(VI) reduction rate, with four times higher catalytic activity compared to commercial Pd/C. The potential applicability of S. tokodaii cells in the recovery of highly catalytic Pd(0) nanoparticles from actual acidic chloride leachate was, thus, suggested.

The superfamily of adenylate-forming enzymes all share a common chemistry. They activate a carboxylate group, on a specific substrate, by catalyzing the formation of a high energy mixed phosphoanhydride-linked nucleoside intermediate. Members of this diverse enzymatic family play key roles in a variety of metabolic pathways and therefore many have been regarded as drug targets. A generic approach to inhibit such enzymes is the use of non-hydrolysable sulfur-based bioisosteres of the adenylate intermediate. Here we compare the activity of compounds containing a sulfamoyl and sulfonamide linker respectively. An improved synthetic strategy was developed to generate inhibitors containing the latter that target isoleucyl- (IleRS) and seryl-tRNA synthetase (SerRS), two structurally distinct representatives of Class I and II aminoacyl-tRNA synthetases (aaRSs). These enzymes attach their respective amino acid to its cognate tRNA and are indispensable for protein translation. Evaluation of the ability of the two similar isosteres to inhibit serRS revealed a remarkable difference, with an almost complete loss of activity for seryl-sulfonamide 15 (SerSoHA) compared to its sulfamoyl analogue (SerSA), while inhibition of IleRS was unaffected. To explain these observations, we have determined a 2.1 Å crystal structure of Klebsiella pneumoniae SerRS in complex with SerSA. Using this structure as a template, modelling of 15 in the active site predicts an unfavourable eclipsed conformation. We extended the same modelling strategy to representative members of the whole adenylate-forming enzyme superfamily, and were able to disclose a new classification system for adenylating enzymes, based on their protein fold. The results suggest that, other than for the structural and functional orthologues of the Class II aaRSs, the O to C substitution within the sulfur-sugar link should generally preserve the inhibitory potency., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems incorporate short DNA fragments from invasive genetic elements into host CRISPR arrays in order to generate host immunity. Recently, we demonstrated that the Csa3a regulator protein triggers CCN protospacer-adjacent motif (PAM)-dependent CRISPR spacer acquisition in the subtype I-A CRISPR-Cas system of Sulfolobus islandicus However, the mechanisms underlying specific protospacer selection and spacer insertion remained unclear. Here, we demonstrate that two Cas4 family proteins (Cas4 and Csa1) have essential roles (i) in recognizing the 5' PAM and 3' nucleotide motif of protospacers and (ii) in determining both the spacer length and its orientation. Furthermore, we identify amino acid residues of the Cas4 proteins that facilitate these functions. Overexpression of the Cas4 and Csa1 proteins, and also that of an archaeal virus-encoded Cas4 protein, resulted in strongly reduced adaptation efficiency, and the former proteins yielded a high incidence of PAM-dependent atypical spacer integration or of PAM-independent spacer integration. We further demonstrated that in plasmid challenge experiments, overexpressed Cas4-mediated defective spacer acquisition in turn potentially enabled targeted DNA to escape subtype I-A CRISPR-Cas interference. In summary, these results define the specific involvement of diverse Cas4 proteins in in vivo CRISPR spacer acquisition. Furthermore, we provide support for an anti-CRISPR role for virus-encoded Cas4 proteins that involves compromising CRISPR-Cas interference activity by hindering spacer acquisition. IMPORTANCE The Cas4 family endonuclease is an essential component of the adaptation module in many variants of CRISPR-Cas adaptive immunity systems. The Crenarchaeota Sulfolobus islandicus REY15A carries two cas4 genes ( cas4 and csa1 ) linked to the CRISPR arrays. Here, we demonstrate that Cas4 and Csa1 are essential to CRISPR spacer acquisition in this organism. Both proteins specify the upstream and downstream conserved nucleotide motifs of the protospacers and define the spacer length and orientation in the acquisition process. Conserved amino acid residues, in addition to those recently reported, were identified to be important for these functions. More importantly, overexpression of the Sulfolobus viral Cas4 abolished spacer acquisition, providing support for an anti-CRISPR role for virus-encoded Cas4 proteins that inhibit spacer acquisition., (Copyright © 2019 American Society for Microbiology.)

Archaea have evolved various strategies in chromosomal organization. While histone homologues exist in most archaeal phyla, Cren7 is a chromatin protein conserved in the Crenarchaeota. Here, we show that Cren7 preferentially binds DNA with AT-rich sequences over that with GC-rich sequences with a binding size of 6~7 bp. Structural studies of Cren7 in complex with either an 18-bp or a 20-bp double-stranded DNA fragment reveal that Cren7 binds to the minor groove of DNA as monomers in a head-to-tail manner. The neighboring Cren7 monomers are located on the opposite sides of the DNA duplex, with each introducing a single-step sharp kink by intercalation of the hydrophobic side chain of Leu28, bending the DNA into an S-shape conformation. A structural model for the chromatin fiber folded by Cren7 was established and verified by the analysis of cross-linked Cren7-DNA complexes by atomic force microscopy. Our results suggest that Cren7 differs significantly from Sul7, another chromatin protein conserved among Sulfolobus species, in both DNA binding and deformation. These data shed significant light on the strategy of chromosomal DNA organization in crenarchaea., (© 2018 John Wiley & Sons Ltd.)

Iron oxide nanoparticles (NPs) are attractive materials for enzyme immobilization and, thanks to their superparamagnetism, can be accessed by remote stimuli. This can be exploited to activate molecules that are not remotely actuable. Here, we demonstrate that thermophilic enzymes chemically linked to NPs can be activated in a "wireless" fashion by an external alternate magnetic field (AMF). To this aim, we have conjugated, with different binding strategies, the thermophilic enzymes α-amylase and l-aspartate oxidase to iron oxide NPs obtaining NP-enzyme systems with activities depending on the different orientations and stretching of the enzymes. Since enzyme activation occurs without a significant rise of the "overall" temperature of the systems, we have speculated a local NP-enzyme heating that does not immediately interest the rest of the solution that remains at relatively low temperature, low enough to allow non-thermophilic enzymes to work together with the NP-conjugated thermophilic enzymes. Nanoactuation of thermophilic enzymes by AMF has potential applications in different fields. Indeed, multi-enzymatic processes with enzymes with different temperature optima could be carried out in the same reaction pot and thermolabile products could be efficiently produced by thermophilic enzymes without suffering for the high temperatures. Moreover, our findings represent a proof of concept of the possibility to achieve a fine-tuning of the enzyme-NP system with the aim to intervene in cell metabolism., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Isocitrate dehydrogenase is a catabolic enzyme that acts during the third step of the tricarboxylic acid cycle. The hypothetical protein ST2166 from the archaeon Sulfolobus tokodaii was isolated and crystallized. It shares high primary structure homology with prokaryotic NADP + -dependent IDHs, suggesting that these enzymes share a common enzymatic mechanism. The crystal structure of ST2166 was determined at 2.0 Å resolution in the apo form, and then the structure of the crystal soaked with NADP + was also determined at 2.4 Å resolution, which contained NADP + bound at the putative active site. Comparisons between the structures of apo and NADP + -bound forms and NADP-IDHs from other prokaryotes suggest that prokaryotic NADP-IDHs recognize their cofactors using conserved Lys335, Tyr336, and Arg386 in ST2166 at the opening cleft before the domain closure.

We showed previously that the Y97N mutant of the ST0452 protein, isolated from Sulfolobus tokodaii , exhibited over 4 times higher N -acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase) activity, compared with that of the wild-type ST0452 protein. We determined the three-dimensional structure of the Y97N protein to explore the detailed mechanism underlying this increased activity. The overall structure was almost identical to that of the wild-type ST0452 protein (PDB ID 2GGO), with residue 97 (Asn) interacting with the O-5 atom of N -acetylglucosamine (GlcNAc) in the complex without metal ions. The same interaction was observed for Escherichia coli GlmU in the absence of metal ions. These observations indicated that the three-dimensional structure of the Y97N protein was not changed by this substitution but the interactions with the substrate were slightly modified, which might cause the activity to increase. The crystal structure of the Y97N protein also showed that positions 146 (Glu) and 80 (Thr) formed interactions with GlcNAc, and an engineering strategy was applied to these residues to increase activity. All proteins substituted at position 146 had drastically decreased activities, whereas several proteins substituted at position 80 showed higher GlcNAc-1-P UTase activity, compared to that of the wild-type protein. The substituted amino acids at positions 80 and 97 might result in optimized interactions with the substrate; therefore, we predicted that the combination of these two substitutions might cooperatively increase GlcNAc-1-P UTase activity. Of the four double mutant ST0452 proteins generated, T80S/Y97N showed 6.5-times-higher activity, compared to that of the wild-type ST0452 protein, revealing that these two substituted residues functioned cooperatively to increase GlcNAc-1-P UTase activity. IMPORTANCE We demonstrated that the enzymatic activity of a thermostable protein was over 4 times higher than that of the wild-type protein following substitution of a single amino acid, without affecting its thermostability. The three-dimensional structure of the improved mutant protein complexed with substrate was determined. The same overall structure and interaction between the substituted residue and the GlcNAc substrate as observed in the well-characterized bacterial enzyme suggested that the substitution of Tyr at position 97 by Asn might slightly change the interaction. This subtle change in the interaction might potentially increase the GlcNAc-1-P UTase activity of the mutant protein. These observations indicated that a drastic change in the structure of a natural thermostable enzyme is not necessary to increase its activity; a subtle change in the interaction with the substrate might be sufficient. Cooperative effects were observed in the appropriate double mutant protein. This work provides useful information for the future engineering of natural enzymes., (Copyright © 2018 American Society for Microbiology.)

Glycosyltrehalose synthase (GTSase) converts the glucosidic bond between the last two glucose residues of amylose from an α-1,4 bond to an α-1,1 bond, generating a nonreducing glycosyl trehaloside, in the first step of the biosynthesis of trehalose. To better understand the structural basis of the catalytic mechanism, the crystal structure of GTSase from the hyperthermophilic archaeon Sulfolobus shibatae DSM5389 (5389-GTSase) has been determined to 2.4 Å resolution by X-ray crystallography. The structure of 5389-GTSase can be divided into five domains. The central domain contains the (β/α) 8 -barrel fold that is conserved as the catalytic domain in the α-amylase family. Three invariant catalytic carboxylic amino acids in the α-amylase family are also found in GTSase at positions Asp241, Glu269 and Asp460 in the catalytic domain. The shape of the catalytic cavity and the pocket size at the bottom of the cavity correspond to the intramolecular transglycosylation mechanism proposed from previous enzymatic studies.

In this work, a new exploitation of the thermostable β-glycosidase from Sulfolobus solfataricus expressed in Saccharomyces cerevisiae to create functional foods for low lactose diets was evaluated. For this purpose, the lactose hydrolysis reaction using immobilized and soluble enzymes was investigated. Activity and stability at different conditions of pH and temperature were tested. The immobilization process had a big impact on the catalysis performance, leading to an enhancement of the enzymatic reaction rate on lactose, as demonstrated by the increasing of 2 and 2.5 folds of K cat and K cat /K M , respectively. Moreover, the maximal activity for the immobilized form was referred at pH 6.5 instead of 7.0, leading to an improvement of the catalytic performance at milk pHs. Although the soluble enzyme was already weakly inhibited by the reaction products, the immobilization further reduced the inhibitory action of glucose increasing the K i from 96.7 to 110.4 mM. Finally, the immobilized enzyme showed high hydrolysis rate in whole milk that yielded 99% of lactose breakdown in 10 and 30 min at 60 and 40°C, respectively. These results support the application of the immobilized β-glycosidase for the development of new functional foods particularly suitable to the alleviation of lactose intolerance., (© 2018 Wiley Periodicals, Inc.)

PINA is a novel ATPase and DNA helicase highly conserved in Archaea, the third domain of life. The PINA from Sulfolobus islandicus (SisPINA) forms a hexameric ring in crystal and solution. The protein is able to promote Holliday junction (HJ) migration and physically and functionally interacts with Hjc, the HJ specific endonuclease. Here, we show that SisPINA has direct physical interaction with Hjm (Hel308a), a helicase presumably targeting replication forks. In vitro biochemical analysis revealed that Hjm, Hjc, and SisPINA are able to coordinate HJ migration and cleavage in a concerted way. Deletion of the carboxyl 13 amino acid residues impaired the interaction between SisPINA and Hjm. Crystal structure analysis showed that the carboxyl 70 amino acid residues fold into a type II KH domain which, in other proteins, functions in binding RNA or ssDNA. The KH domain not only mediates the interactions of PINA with Hjm and Hjc but also regulates the hexameric assembly of PINA. Our results collectively suggest that SisPINA, Hjm and Hjc work together to function in replication fork regression, HJ formation and HJ cleavage.

The DNA nuclease gene ST2109 has been cloned from the hyperthermophilic archaeon Sulfolobus tokodaii and expressed in Escherichia coli. The recombinant protein StoNurA has been purified to homogeneity by affinity chromatography and gel filtration chromatography. Biochemical analyses demonstrated that StoNurA exhibited DNA binding and 5'-3' exonuclease activities towards ssDNA and dsDNA. The temperature and pH optima of StoNurA were determined to be 65 °C and 8.0, respectively. The activity of StoNurA was found to be dependent of Mn2+, and its half-life of heat inactivation at 100 °C was 5 min. Gel filtration chromatography revealed that StoNurA could form dimers in solution. Pull-down assays also showed that StoNurA physically interacted with a DNA helicase (StoHerA). Our data suggest that NurA may play a key functional role in the processing of DNA recombinational repair.

The study of evolution is important to understand biological phenomena. During evolutionary processes, genetic changes confer amino acid substitutions in proteins, resulting in new or improved functions. Unfortunately, most mutations destabilize proteins. Thus, protein stability is a significant factor in evolution; however, its role remains unclear. Here, we simply and directly explored the association between protein activity and stability in random mutant libraries to elucidate the role of protein stability in evolutionary processes. In the first random mutation of an esterase from Sulfolobus tokodaii, approximately 20% of the variants displayed higher activity than wild-type protein (i.e., 20% evolvability). During evolutionary processes, the evolvability depended on the stability of template proteins, indicating that protein evolution is potentially governed by protein stability. Furthermore, decreased activity could be recovered during evolution by maintaining the stability of variants. The results suggest that protein sequence space for its evolution is able to expand during nearly neutral evolution where mutations are slightly deleterious for activity but rarely fatal for stability. Molecular evolution is a crucial phenomenon that has continued since the birth of life on earth, and mechanism underlying it is simple; therefore, this could be demonstrated by our simple experiments. These findings also can be applied to protein engineering.

Most organisms, from Bacteria to Eukarya , synthesize UDP- N -acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP- N -acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea , the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii , predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii , and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic pathways. IMPORTANCE In this work, a novel protein capable of directly converting glucosamine-6-phosphate to galactosamine-6-phosphate was successfully purified from a cell extract of the thermophilic crenarchaeon Sulfolobus tokodaii Confirmation of this novel activity using the recombinant protein indicates that S. tokodaii possesses a novel UDP-GalNAc biosynthetic pathway derived from glucosamine-6-phosphate. The distributions of this and related genes indicate the presence of three different types of UDP-GalNAc biosynthetic pathways: a direct pathway using a novel enzyme and two conversion pathways from UDP-GlcNAc using known enzymes. Additionally, Crenarchaeota species lacking all three pathways were found, predicting the presence of one more unknown pathway. Identification of these novel proteins and pathways provides important insights into the evolution of nucleotide sugar biosynthesis, as well as being potentially important industrially., (Copyright © 2018 American Society for Microbiology.)

Phytoene (C 40 H 64 ) is an isoprenoid and a precursor of various carotenoids which are of industrial value. Archaea can be considered to exhibit a relatively large capacity to produce isoprenoids, as they are components of their membrane lipids. Here, we aimed to produce isoprenoids such as phytoene in the hyperthermophilic archaeon Thermococcus kodakarensis. T. kodakarensis harbors a prenyltransferase gene involved in the biosynthesis of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which are precursors of squalene and phytoene, respectively. However, homologs of squalene synthase and phytoene synthase, which catalyze their condensation reactions, are not found on the genome. Therefore, a squalene/phytoene synthase homolog from an acidothermophilic archaeon Sulfolobus acidocaldarius, Saci_1734, was introduced into the T. kodakarensis chromosome under the control of a strong promoter. Production of the Saci_1734 protein was confirmed in this strain, and the generation of phytoene was detected (0.08-0.75 mg L -1 medium). We then carried out genetic engineering in order to increase the phytoene production yield. Disruption of an acetyl-CoA synthetase I gene involved in hydrolyzing acetyl-CoA, the precursor of phytoene, together with the introduction of a second copy of Saci_1734 led to a 3.4-fold enhancement in phytoene production.

Much remains to be understood about the kinetics and thermodynamics of DNA helicase binding and activity. Here, we utilize probe-modified DNA monolayers on multiplexed gold electrodes as a sensitive recognition element and morphologically responsive transducer of helicase-DNA interactions. The electrochemical signals from these devices are highly sensitive to structural distortion of the DNA produced by the helicases. We used this DNA electrochemistry to distinguish the details of the DNA interactions of three distinct XPB helicases, which belong to the superfamily-2 of helicases. Clear changes in DNA melting temperature and duplex stability were observed upon helicase binding, shifts that could not be observed with conventional UV-visible absorption measurements. Binding dissociation constants were estimated in the range from 10 to 50 nM and correlated with observations of activity. ATP-stimulated DNA unwinding activity was also followed, revealing exponential time scales and distinct time constants associated with conventional and molecular wrench modes of operation further confirmed by crystal structures. These devices thus provide a sensitive measure of the structural thermodynamics and kinetics of helicase-DNA interactions.

CRISPR-Cas systems protect prokaryotes against invading viruses and plasmids. The system is associated with a large number of Cas accessory proteins among which many contain a CARF (CRISPR-associated Rossmann fold) domain implicated in ligand binding and a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) nuclease domain. Here, such a dual domain protein, i.e. the Sulfolobus islandicus Csx1 (SisCsx1) was characterized. The enzyme exhibited metal-independent single-strand specific ribonuclease activity. In fact, SisCsx1 showed a basal RNase activity in the absence of ligand; upon the binding of an RNA ligand carrying four continuous adenosines at the 3'-end (3'-tetra-rA), the activated SisCsx1 degraded RNA substrate with a much higher turnover rate. Amino acid substitution mutants of SisCsx1 were obtained, and characterization of these mutant proteins showed that the CARF domain of the enzyme is responsible for binding to 3'-tetra-rA and the ligand binding strongly activates RNA cleavage by the HEPN domain. Since RNA polyadenylation is an important step in RNA decay in prokaryotes, and poly(A) RNAs can activate CARF domain proteins, the poly(A) RNA may function as an important signal in the cellular responses to viral infection and environmental stimuli, leading to degradation of both viral and host transcripts and eventually to cell dormancy or cell death., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Enzymes from thermophilic and hyper-thermophilic organisms have an intrinsic high stability. Understanding the mechanisms behind their high stability will be important knowledge for the engineering of novel enzymes with high stability. Lysine methylation of proteins is prevalent in Sulfolobus, a genus of hyperthermophilic and acidophilic archaea. Both unspecific and temperature dependent lysine methylations are seen, but the significance of this post-translational modification has not been investigated. Here, we test the effect of eliminating in vivo lysine methylation on the stability of an esterase (EstA). The enzyme was purified from the native host S. islandicus as well as expressed as a recombinant protein in E. coli, a mesophilic host that does not code for any machinery for in vivo lysine methylation. We find that lysine mono methylation indeed has a positive effect on the stability of EstA, but the effect is small. The effect of the lysine methylation on protein stability is secondary to that of protein expression in E. coli, as the E. coli recombinant enzyme is compromised both on stability and activity. We conclude that these differences are not attributed to any covalent difference between the protein expressed in hyperthermophilic versus mesophilic hosts., (© 2017 The Protein Society.)

AP endonuclease recognizes and cleaves apurinic/apyrimidinic (AP) sites and plays a critical role in base excision repair. Many ExoIII and EndoIV family AP endonucleases have been characterized both biochemically and structurally in Eukaryote and Bacteria. However, relatively fewer have been studied in Euryarchaeota and there is no such report on an AP endonuclease from Crenarchaeota. Here we report, for the first time, the crystal structure of a crenarchaeal ExoIII AP endonuclease, SisExoIII, from Sulfolobus islandicus REY15A. SisExoIII comprises a two-layer core formed by 10 β-sheets and a shell formed by 9 surrounding α-helices. A disulfide bond connecting β8 and β9 is formed by Cys142 and Cys215. This intra-molecular linkage is conserved among crenarchaeal ExoIII homologs and site-directed mutagenesis revealed that it endows the protein with thermostability, however, disruption of the disulfide bond only has a slight effect on the AP endonuclease activity. We also observed that several key residues within the catalytic center including conserved Glu35 and Asn9 show different conformation compared with known ExoIII proteins and form various intra-molecular salt bridges. The protein possesses three putative DNA binding loops with higher flexibility and hydrophobicity than those of ExoIIIs from other organisms. These features may result in low AP endonuclease activity and defect of exonuclease activity of SisExoIII. The study has deepened our understanding in the structural basis of crenarchaeal ExoIII catalysis and clarified a role of the disulfide bond in maintaining protein thermostability., (Copyright © 2017. Published by Elsevier Inc.)

Thymidylate kinase (TMK) is a key enzyme for the synthesis of DNA, making it an important target for the development of anticancer, antibacterial, and antiparasitic drugs. TMK homologs exhibit significant variations in sequence, residue conformation, substrate specificity, and oligomerization mode. However, the influence of sequence evolution and conformational dynamics on its quaternary structure and function has not been studied before. Based on extensive sequence and structure analyses, our study detected several non-conserved residues which are linked by co-evolution and are implicated in the observed variations in flexibility, oligomeric assembly, and substrate specificity among the homologs. These lead to differences in the pattern of interactions at the active site in TMKs of different specificity. The method was further tested on TMK from Sulfolobus tokodaii (StTMK) which has substantial differences in sequence and structure compared to other TMKs. Our analyses pointed to a more flexible dTMP-binding site in StTMK compared to the other homologs. Binding assays proved that the protein can accommodate both purine and pyrimidine nucleotides at the dTMP binding site with comparable affinity. Additionally, the residues responsible for the narrow specificity of Brugia malayi TMK, whose three-dimensional structure is unavailable, were detected. Our study provides a residue-level understanding of the differences observed among TMK homologs in previous experiments. It also illustrates the correlation among sequence evolution, conformational dynamics, oligomerization mode, and substrate recognition in TMKs and detects co-evolving residues that affect binding, which should be taken into account while designing novel inhibitors.

Reverse gyrase introduces positive supercoils to circular DNA and is implicated in genome stability maintenance in thermophiles. The extremely thermophilic crenarchaeon Sulfolobus encodes two reverse gyrase proteins, TopR1 (topoisomerase reverse gyrase 1) and TopR2, whose functions in thermophilic life remain to be demonstrated. Here, we investigated the roles of TopR1 in genome stability maintenance in S. islandicus in response to the treatment of methyl methanesulfonate (MMS), a DNA alkylation agent. Lethal MMS treatment induced two successive events: massive chromosomal DNA backbone breakage and subsequent DNA degradation. The former occurred immediately after drug treatment, leading to chromosomal DNA degradation that concurred with TopR1 degradation, followed by chromatin protein degradation and DNA-less cell formation. To gain a further insight into TopR1 function, the expression of the enzyme was reduced in S. islandicus cells using a CRISPR-mediated mRNA interference approach (CRISPRi) in which topR1 mRNAs were targeted for degradation by endogenous III-B CRISPR-Cas systems. We found that the TopR1 level was reduced in the S. islandicus CRISPRi cells and that the cells underwent accelerated genomic DNA degradation during MMS treatment, accompanied by a higher rate of cell death. Taken together, these results indicate that TopR1 probably facilitates genome integrity maintenance by protecting DNA breaks from thermo-degradation in vivo., Competing Interests: The authors declare no conflict of interest.

In this paper, the dephosphorylation mechanism of FBP to F6P catalyzed by the Fructose-1, 6-bisphosphatase (St-Fbp) from Sulfolobus tokodaii was studied using quantum mechanical/molecular mechanical (QM/MM) approach. Based on the experimental results, total five possible catalytic mechanisms (path1-path4') were designed. The most possible dephosphorylation reaction follows a two-step mechanism (path2): a dephosphorylation process (with D12 being an base of W6 and residue K133 being the proton donor of the linking FBP:O4) and a proton exchange process (between K133 and the water W1). Furthermore, the three-step of path4 is also possible: a dephosphorylation process (with D54 being the base of W6 and residue K133 being the proton donor of the linking FBP:O4) and two proton exchange processes (first between residues D54 and D12 then between K133 and the water W1). The relative low energy of this pathway suggests that D54 might also be a base except D12. Our calculations indicate that K133 is the preferred proton donor during the breaking of the phosphate bond O4-P1, with the W1 being an alternative proton donor to access to a more stable product. Findings here give a new insight into the understanding of catalytic mechanism of FBPase., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Complete genome analysis of the thermoacidophilic Archaeon Sulfolobus tokodaii strain 7 revealed two open reading frames (ORF), namely, ST0926 and ST0927. These ORFs are interrupted by two putative insertions and encode for the N- and C-terminal fragments, respectively, of a putative Sulfolobus sp. maltooligosyltrehalose trehalohydrolase (StMTHase). Two specific deletion mutations, designed on the basis of sequence alignments of the adjacent ORFs and the published Sulfolobus sp. MTHases, allowed soluble expression in Escherichia coli of an active acidic and thermophilic enzyme. The purified enzyme exhibited a maximum amylolytic activity at 70 °C and pH 5.0, which resembled the optimal conditions of the Sulfolobus homologs. Furthermore, we report that these ORFs are actively co-transcribed in vivo, and we confirm the presence of insertions between them at the cDNA level. However, immunization and western blot experiments demonstrated no expression of ST0926 or the putative full-length StMTHase in vivo, indicating that they might exist as nonfunctional pseudogenes.

Holliday junction (HJ) is a hallmark intermediate in DNA recombination and must be processed by dissolution (for double HJ) or resolution to ensure genome stability. Although HJ resolvases have been identified in all domains of life, there is a long-standing effort to search in prokaryotes and eukarya for proteins promoting HJ migration. Here, we report the structural and functional characterization of a novel ATPase, Sulfolobus islandicusPilT N-terminal-domain-containing ATPase (SisPINA), encoded by the gene adjacent to the resolvase Hjc coding gene. PINA is conserved in archaea and vital for S. islandicus viability. Purified SisPINA forms hexameric rings in the crystalline state and in solution, similar to the HJ migration helicase RuvB in Gram-negative bacteria. Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration. Further studies reveal that SisPINA interacts with SisHjc and coordinates HJ migration and cleavage., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Thymidylate kinase (TMK) is a key enzyme that plays an important role in DNA synthesis. Therefore, it serves as an attractive therapeutic target for the development of antibacterial, antiparasitic and anticancer drugs. Herein, we report the biochemical characterization and crystal structure determination of thymidylate kinase from a hyperthermophilic organism Sulfolobus tokodaii (StTMK) in its apo and ADP-bound forms. Our study describes the first three-dimensional structure of an archaeal TMK. StTMK is a thermostable enzyme with optimum activity at 80°C. Despite the overall similarity to homologous TMKs, StTMK structures revealed several residue substitutions at the active site. However, enzyme assays demonstrated specificity to its natural substrates ATP and dTMP. Analysis of the structures also revealed multiple conformational states of Arg93 which is located at the reaction centre and is a part of the highly conserved DRX motif. Only one of these states was found to be suitable for the proper positioning of the α-phosphate group of dTMP at the active site. Computational alanine scanning and MM/PBSA binding energy calculation revealed the importance of Arg93 side chain in substrate binding. Subsequent site directed mutagenesis at this position to an Ala resulted in the loss of activity. Thus, the computational and biochemical studies reveal the importance of Arg93 for enzyme function, while the different conformational states of Arg93 observed in the structural studies imply its regulatory role in the catalytically competent placement of dTMP., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the formation of cystathionine from an L-homoserine derivative and L-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P2 1 ; unit-cell parameters a = 58.4, b = 149.3, c = 90.2 Å, β = 108.9°) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C222 1 ; unit-cell parameters a = 117.7, b = 117.8, c = 251.3 Å) and in form II' at 313 K (space group C222 1 ; unit-cell parameters a = 107.5, b = 127.7, c = 251.1 Å) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 Å resolution for forms I, II and II', respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II', suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

As the first three-dimensional structure of the two-subunit type 2-oxoacid:ferredoxin oxidoreductases (OFOR) from archaea, we solved the crystal structures of STK_23000/STK_22980 (StOFOR1) and STK_24350/STK_24330 (StOFOR2) from Sulfolobus tokodaii. They showed similar overall structures, consisting of two a- and b-subunit heterodimers containing thiamin pyrophosphate (TPP) cofactor and [4Fe-4S] cluster, but lack an intramolecular ferredoxin domain. Unlike other OFORs, StOFORs can utilize both pyruvate and 2-oxoglutarate, playing a key role in the central metabolism. In the structure of StOFOR2 in unreacted pyruvate complex form, carboxylate group of pyruvate is recognized by Arg344 and Thr257 from the a-subunit, which are conserved in pyruvate:ferredoxin oxidoreductase from Desulfovbrio africanus (DaPFOR). In the structure of StOFOR1 co-crystallized with 2-oxobutyrate, electron density corresponding to a 1-hydroxypropyl group (post-decarboxylation state) was observed at the thiazole ring of TPP. The binding pockets of the StOFORs surrounding the methyl or propyl group of the ligands are wider than that of DaPFOR. Mutational analyses indicated that several residues were responsible for the broad 2-oxoacid specificity of StOFORs. We also constructed a possible complex structural model by placing a Zn(2+)-containing dicluster ferredoxin of S. tokodaii into the large pocket of StOFOR2, providing insight into the electron transfer between the two redox proteins.

AP endonuclease cleaves the phosphodiester bond 5'- to the AP (apurinic or apyrimidinic) sites and is one of the major enzymes involved in base excision repair. So far, the properties of several archaeal AP endonuclease homologues have been characterized in vitro, but little is known about their functions in vivo. Herein, we report on the biochemical and genetic analysis of two AP endonucleases, SisExoIII and SisEndoIV, from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A. Both SisExoIII and SisEndoIV exhibit AP endonuclease activity, but neither of them has 3'-5' exonuclease activity. SisExoIII and SisEndoIV have similar K M values on the substrate containing an AP site, but the latter cleaves the AP substrate at a dramatically higher catalytic rate than the former. Unlike other AP endonucleases identified in archaea, SisExoIII and SisEndoIV do not exhibit any cleavage activity on DNA having oxidative damage (8-oxo-dG) or uracil. Genetic analysis revealed that neither gene is essential for cell viability, and the growth of ∆SiRe_2666 (endoIV), ∆SiRe_0100 (exoIII), and ∆SiRe_0100∆SiRe_2666 is not affected under normal growth conditions. However, ∆SiRe_2666 exhibits higher sensitivity to the alkylating agent methyl methanesulfonate (MMS) than ∆SiRe_0100. Over-expression of SiRe_0100 can partially complement the sensitivity of ∆SiRe_2666 to MMS, suggesting a backup role of SisExoIII in AP site processing in vivo. Intriguingly, over-expression of SisEndoIV renders the strain more sensitive to MMS than the control. Taken together, we conclude that SisEndoIV, but not SisExoIII, is the main AP endonuclease that participates directly in base excision repair in S. islandicus.

The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii were determined and their structural characteristics were analyzed. For PurS from T. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.

DExD/H-box helicases represent the largest family of helicases. They belong to superfamily 2 helicases and participate in nucleotide metabolism, ribosome biogenesis, and nucleocytoplasmic transport. The biochemical properties and structures of some DExD/H-box helicases in the archaea have been documented, but many of them have not been characterized; and reports on in vivo functional analyses are limited. In this study, we attempted gene knockout of 8 putative DExD/H-box helicases in Sulfolobus islandicus REY15A and obtained two deletion mutants, SiRe_0681 and SiRe_1605. We determined that ΔSiRe_0681 grew faster than wild type cells in the presence of methyl methanesulfonate (MMS). Flow cytometry analysis showed that this strain had fewer G1/S phase cells than the wild type, and the genes coding for cell division proteins were up-regulated. The stain ΔSiRe_1605 was more sensitive to MMS than the wild type cell, and many nucleotide metabolism and DNA repair enzymes were found to be down-regulated. Intriguingly, deletion of either gene led to silencing simultaneously of over 80 genes located at a specific region. This study provides a novel insight into the in vivo functions of predicted DExD/H-box family helicases in the archaea.

In archaea, the HEL308 homolog Hel308a (or Hjm) is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is limited. Herein, a structure-based functional analysis of Hjm was performed in the genetically tractable hyperthermophilic archaeon, Sulfolobus islandicus. Results showed that domain V and residues within it, which affect Hjm activity and regulation, are essential and that the domain V-truncated mutants and site-directed mutants within domain V cannot complement hjm chromosomal deletion. Chromosomal hjm deletion can be complemented by ectopic expression of hjm under the control of its native promoter but not an artificial arabinose promoter. Cellular Hjm levels are kept constant under ultraviolet (UV) and methyl methanesulfonate (MMS) treatment conditions in a strain carrying a plasmid to induce Hjm overexpression. These results suggest that Hjm expression and activity are tightly controlled, probably at the translational level., (Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: Sulfolobus islandicus serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in S. islandicus to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in S. islandicus M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by M164_0158 (apt). The application of the apt gene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in S. islandicus M.16.4 at the apt locus. Moreover, the general conservation of apt genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the apt locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon S. islandicus, Importance: Currently, the pyrEF/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most Sulfolobus mutants constructed by the research community were derived from genetic hosts lacking the pyrEF genes, the pyrEF/5-FOA system is no longer available for use in forward mutation assays. Demonstration of the apt/6-MP counterselection system for the Sulfolobus model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a pyrEF-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among S. islandicus strains., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5' to 3' helicase with an essential iron-sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase from Thermoplasma acidophilum has been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD from Sulfolobus acidocaldiarius that lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

α-Glucose 1-phosphate (G1P) is synthesized from 5% (w/v) corn starch and 1 M phosphate mediated by α-glucan phosphorylase (αGP) from the thermophilic bacterium Thermotoga maritima at pH 7.2 and 70 °C. To increase G1P yield from corn starch containing branched amylopectin, a hyper-thermostable isoamylase from Sulfolobus tokodaii was added for simultaneous starch gelatinization and starch-debranching hydrolysis at 85 °C and pH 5.5 before αGP use. The pretreatment of isoamylase increased G1P titer from 120 mM to 170 mM. To increase maltose and maltotriose utilization, the third thermostable enzyme, 4-glucanotransferase (4GT) from Thermococcus litoralis, was added during the late stage of G1P biotransformation, further increasing G1P titer to 200 mM. This titer is the highest G1P level obtained on starch or its derived products (maltodextrin and soluble starch). This study suggests that in vitro multienzyme biotransformation has an advantage of great engineering flexibility in terms of space and time compared with microbial fermentation.

The SPO11 protein catalyzes the formation of meiotic DNA double strand breaks (DSBs) and is homologous to the A subunit of an archaeal topoisomerase (topo VI). Topo VI are heterotetrameric enzymes comprising two A and two B subunits; however, no topo VIB involved in meiotic recombination had been identified. We characterized a structural homolog of the archaeal topo VIB subunit [meiotic topoisomerase VIB-like (MTOPVIB)], which is essential for meiotic DSB formation. It forms a complex with the two Arabidopsis thaliana SPO11 orthologs required for meiotic DSB formation (SPO11-1 and SPO11-2) and is absolutely required for the formation of the SPO11-1/SPO11-2 heterodimer. These findings suggest that the catalytic core complex responsible for meiotic DSB formation in eukaryotes adopts a topo VI-like structure., (Copyright © 2016, American Association for the Advancement of Science.)

Archaea use glycolytic pathways distinct from those found in bacteria and eukaryotes, where unique enzymes catalyze each reaction step. In this study, we isolated three isozymes of glyceraldehyde oxidoreductase (GAOR1, GAOR2 and GAOR3) from the thermoacidophilic archaeon Sulfolobus tokodaii. GAOR1-3 belong to the xanthine oxidoreductase superfamily, and are composed of a molybdo-pyranopterin subunit (L), a flavin subunit (M), and an iron-sulfur subunit (S), forming an LMS hetero-trimer unit. We found that GAOR1 is a tetramer of the STK17810/STK17830/STK17820 hetero-trimer, GAOR2 is a dimer of the STK23390/STK05620/STK05610 hetero-trimer, and GAOR3 is the STK24840/STK05620/STK05610 hetero-trimer. GAOR1-3 exhibited diverse substrate specificities for their electron donors and acceptors, due to their different L-subunits, and probably participate in the non-phosphorylative Entner-Doudoroff glycolytic pathway. We determined the crystal structure of GAOR2, as the first three-dimensional structure of an archaeal molybdenum-containing hydroxylase, to obtain structural insights into their substrate specificities and subunit assemblies. The gene arrangement and the crystal structure suggested that the M/S-complex serves as a structural scaffold for the binding of the L-subunit, to construct the three enzymes with different specificities. Collectively, our findings illustrate a novel principle of a prokaryotic multicomponent isozyme system.

Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins., (Copyright © 2016 Elsevier Inc. All rights reserved.)