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

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

Enhancing the Catalytic Activity of Thermo-Asparaginase from Thermococcus sibiricus by a Double Mesophilic-like Mutation in the Substrate-Binding Region.

L-asparaginases (L-ASNases) of microbial origin are the mainstay of blood cancer treatment. Numerous attempts have been performed for genetic improvement of the main properties of these enzymes. The substrate-binding Ser residue is highly conserved in L-ASNases regardless of their origin or type. However, the residues adjacent to the substrate-binding Ser differ between mesophilic and thermophilic L-ASNases. Based on our suggestion that the triad, including substrate-binding Ser, either GSQ for meso-ASNase or DST for thermo-ASNase, is tuned for efficient substrate binding, we constructed a double mutant of thermophilic L-ASNase from Thermococcus sibiricus (TsA) with a mesophilic-like GSQ combination. In this study, the conjoint substitution of two residues adjacent to the substrate-binding Ser55 resulted in a significant increase in the activity of the double mutant, reaching 240% of the wild-type enzyme activity at the optimum temperature of 90 °C. The mesophilic-like GSQ combination in the rigid structure of the thermophilic L-ASNase appears to be more efficient in balancing substrate binding and conformational flexibility of the enzyme. Along with increased activity, the TsA D54G/T56Q double mutant exhibited enhanced cytotoxic activity against cancer cell lines with IC90 values from 2.8- to 7.4-fold lower than that of the wild-type enzyme.

Biochemical and Functional Characterization of an Iron-Containing Alcohol Dehydrogenase from Thermococcus barophilus Ch5.

Two iron-containing alcohol dehydrogenases (ADHs) are encoded in the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba ADH641 and Tba ADH547). In our previous publication, we reported biochemical characteristics and catalytic mechanism of Tba ADH547. Herein, we present evidence that Tba ADH641 possesses two activities for ethanol oxidization and acetaldehyde reduction at high temperature, capable of using NAD(H) and NADP(H) as coenzyme. Biochemical data show that Tba ADH641 possesses optimal reaction temperature, thermostability, divalent ion requirement, and substrate specificity distinct from Tba ADH547 and other iron-containing ADH homologues. However, Tba ADH641 and Tba ADH547 display same optimal reaction pH. Kinetic analyses demonstrate that Tba ADH641 displays higher catalytic efficiency for acetaldehyde reduction than that for ethanol oxidation, which is consistent with Tba ADH547. Mutational data demonstrate that residues D115, K118, E159, D190, and E215 in Tba ADH641, which has not been described to date, are necessary for enzyme activity, thus augmenting our understanding on catalytic mechanism of iron-containing ADH. Overall, our work demonstrates that Tba ADH641 is an iron-containing ADH with novel features, which is distinct from Tba ADH547, thus providing a potential biocatalyst for biotransformation reaction.

PCNA from Thermococcus gammatolerans: A protein involved in chromosomal DNA metabolism intrinsically resistant at high levels of ionizing radiation.

Proliferating cell nuclear antigen (PCNA) is an essential protein for cell viability in archaea and eukarya, since it is involved in DNA replication and repair. In order to obtain insights regarding the characteristics that confer radioresistance, the structural study of the PCNA from Thermococcus gammatolerans (PCNATg ) in a gradient of ionizing radiation by X-ray crystallography was carried out, together with a bioinformatic analysis of homotrimeric PCNA structures, their sequences, and their molecular interactions. The results obtained from the datasets and the accumulated radiation dose for the last collection from three crystals revealed moderate and localized damage, since even with the loss of resolution, the electron density map corresponding to the last collection allowed to build the whole structure. Attempting to understand this behavior, multiple sequence alignments, and structural superpositions were performed, revealing that PCNA is a protein with a poorly conserved sequence, but with a highly conserved structure. The PCNATg presented the highest percentage of charged residues, mostly negatively charged, with a proportion of glutamate more than double aspartate, lack of cysteines and tryptophan, besides a high number of salt bridges. The structural study by X-ray crystallography reveals that the PCNATg has the intrinsic ability to resist high levels of ionizing radiation, and the bioinformatic analysis suggests that molecular evolution selected a particular composition of amino acid residues, and their consequent network of synergistic interactions for extreme conditions, as a collateral effect, conferring radioresistance to a protein involved in the chromosomal DNA metabolism of a radioresistant microorganism.

Structural basis for peptide recognition by archaeal oligopeptide permease A.

Oligopeptide permease A (OppA) plays an important role in the nutrition of cells and various signaling processes. In archaea, OppA is a major protein present in membrane vesicles of Thermococcales. Because there being no crystal structures of archaeal OppAs determined to date, we report the crystal structure of archaeal OppA from Thermococcus kodakaraensis (TkOppA) at 2.3 Å resolution by the single-wavelength anomalous dispersion method. TkOppA consists of three domains similarly to bacterial OppAs, and the inserted regions not present in bacterial OppAs are at the periphery of the core region. An endogenous pentapeptide was bound in the pocket of domains I and III of TkOppA by hydrogen bonds of main-chain atoms of the peptide and hydrophobic interactions. No hydrogen bonds of side-chain atoms of the peptide were observed; thus, TkOppA may have low peptide selectivity but some preference for residues 2 and 3. TkOppA has a relatively large pocket and can bind a nonapeptide; therefore, it is suitable for the binding of large peptides similarly to OppAs of Gram-positive bacteria.

Characterization of gluten-degrading prolyl endoprotease from Thermococcus kodakarensis.

There is increasing interest in gluten-degrading enzymes for use during food and drink processing. The industrially available enzymes usually work best at low to ambient temperatures. However, food manufacturing is often conducted at higher temperatures. Therefore, thermostable gluten-degrading enzymes are of great interest. We have identified a new thermostable gluten-degrading proline-specific prolyl endoprotease from the archaea Thermococcus kodakarensis. We then cloned and expressed it in Escherichia coli. The prolyl endoprotease was found to have a size of 70.1 kDa. The synthetic dipeptide Z-Gly-Pro-p-nitroanilide was used to characterize the prolyl endoprotease and it had maximum activity at pH 7 and 77°C. The Vmax, Km and kcat values of the purified prolyl endoprotease were calculated to be 3.14 mM/s, 1.10 mM and 54 s-1, respectively. When the immunogenic gluten peptides PQPQLPYPQPQLPY (α-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein) were used as substrates, the prolyl endoprotease was able to degrade these. Furthermore, gluten in wort was reduced when the prolyl endoprotease was used during mashing of barley malt. The discoveries open up new food processing possibilities and further the understanding of proline-specific protease diversity.

Genomics and simulated laboratory studies reveal Thermococcus sp. 101C5 as a novel hyperthermophilic archaeon possessing a specialized metabolic arsenal for enhanced oil recovery.

Laboratory evaluation of hyperthermophiles with the potential for Enhanced Oil Recovery (EOR) is often hampered by the difficulties in replicating the in situ growth conditions in the laboratory. In the present investigation, genome analysis was used to gain insights into the metabolic potential of a hyperthermophile to mobilize the residual oil from depleting high-temperature oil reservoirs. Here, we report the 1.9 Mb draft genome sequence of a hyperthermophilic anaerobic archaeon, Thermococcus sp. 101C5, with a GC content of 44%, isolated from a high-temperature oil reservoir of Gujarat, India. 101C5 possessed the genetic arsenal required for adaptation to harsh oil reservoir conditions, such as various heat shock proteins for thermo-adaptation, Trk potassium uptake system proteins for osmo-adaptation, and superoxide reductases against oxidative stress. Microbial Enhanced Oil Recovery (MEOR) potential of the strain was established by ascertaining the presence of genes encoding enzymes involved in the production of the metabolites such as hydrogen, bio-emulsifier, acetate, exopolysaccharide, etc. Production of these metabolites which pressurize the reservoir, emulsify the crude oil, lower the viscosity and reduce the drag, thus facilitating mobilization of the residual oil was experimentally confirmed. Also, the presence of crude oil degradative genes highlighted the ability of the strain to mobilize heavy residual oil, which was confirmed under simulated conditions in sand-pack studies. The obtained results demonstrated additional oil recoveries of 42.1% and 56.5% at 96 °C and 101 °C, respectively, by the strain 101C5, illustrating its potential for application in high-temperature oil reservoirs. To our best knowledge, this is the first report of genome analysis of any microbe assessed for its suitability for MEOR from the high-temperature oil reservoir.

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application.

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters KM and Vmax for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0-6 M and displays no significant changes of the activity in the presence of metal ions Ni2+, Cu2+, Mg2+, Zn2+ and Ca2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe3+. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.

Thermostable adenosine 5'-monophosphate phosphorylase from Thermococcus kodakarensis forms catalytically active inclusion bodies.

Catalytically active inclusion bodies (CatIBs) produced in Escherichia coli are an interesting but currently underexplored strategy for enzyme immobilization. They can be purified easily and used directly as stable and reusable heterogenous catalysts. However, very few examples of CatIBs that are naturally formed during heterologous expression have been reported so far. Previous studies have revealed that the adenosine 5'-monophosphate phosphorylase of Thermococcus kodakarensis (TkAMPpase) forms large soluble multimers with high thermal stability. Herein, we show that heat treatment of soluble protein from crude extract induces aggregation of active protein which phosphorolyse all natural 5'-mononucleotides. Additionally, inclusion bodies formed during the expression in E. coli were found to be similarly active with 2-6 folds higher specific activity compared to these heat-induced aggregates. Interestingly, differences in the substrate preference were observed. These results show that the recombinant thermostable TkAMPpase is one of rare examples of naturally formed CatIBs.

Thermococcus camini sp. nov., a hyperthermophilic and piezophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Atlantic Ridge.

A coccoid-shaped, strictly anaerobic, hyperthermophilic and piezophilic organoheterotrophic archaeon, strain Iri35cT, was isolated from a hydrothermal chimney rock sample collected at a depth of 2300 m at the Mid-Atlantic Ridge (Rainbow vent field). Cells of strain Iri35cT grew at NaCl concentrations ranging from 1-5 % (w/v) (optimum 2.0 %), from pH 5.0 to 9.0 (optimum 7.0-7.5), at temperatures between 50 and 90 °C (optimum 75-80 °C) and at pressures from 0.1 to at least 50 MPa (optimum: 10-30 MPa). The novel isolate grew on complex organic substrates, such as yeast extract, tryptone, peptone or beef extract, preferentially in the presence of elemental sulphur or l-cystine; however, these molecules were not necessary for growth. Its genomic DNA G+C content was 54.63 mol%. The genome has been annotated and the metabolic predictions are in accordance with the metabolic characteristics of the strain and of Thermococcales in general. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated ribosomal protein sequences showed that strain Iri35cT belongs to the genus Thermococcus, and is closer to the species T. celericrescens and T. siculi. Average nucleotide identity scores and in silico DNA-DNA hybridization values between the genome of strain Iri35cT and the genomes of the type species of the genus Thermococcus were below the species delineation threshold. Therefore, and considering the phenotypic data presented, strain Iri35cT is suggested to represent a novel species, for which the name Thermococcus camini sp. nov. is proposed, with the type strain Iri35cT (=UBOCC M-2026T=DSM 111003T).

Thermococcus henrietii sp. nov., a novel extreme thermophilic and piezophilic sulfur-reducing archaeon isolated from a deep-sea hydrothermal chimney.

A novel extreme thermophilic and piezophilic chemoorganoheterotrophic archaeon, strain EXT12cT, was isolated from a hydrothermal chimney sample collected at a depth of 2496 m at the East Pacific Rise 9° N. Cells were strictly anaerobic, motile cocci. The strain grew at NaCl concentrations ranging from 1 to 5 % (w/v; optimum, 2.0%), from pH 6.0 to 7.5 (optimum, pH 6.5-7.0), at temperatures between 60 and 95 °C (optimum, 80-85 °C), and at pressures from 0.1 to at least 50 MPa (optimum, 30 MPa). Strain EXT12cT grew chemoorganoheterotrophically on complex proteinaceous substrates. Its growth was highly stimulated by the presence of elemental sulphur or l-cystine, which were reduced to hydrogen sulfide. The DNA G+C content was 54.58 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated ribosomal protein sequences showed that strain EXT12cT falls into the genus Thermococcus and is most closely related to Thermococcus nautili strain 30-1T. Overall genome relatedness index analyses (average nucleotide identity scores and in silico DNA-DNA hybridizations) showed a sufficient genomic distance between the new genome and the ones of the Thermococcus type strains for the delineation of a new species. On the basis of genotypic and phenotypic data, strain EXT12cT is considered to represent a novel species, for which the name Thermococcus henrietii sp. nov. is proposed, with the type strain EXT12cT (=UBOCC M-2417T=DSM 111004T).

ADP-dependent glucose/glucosamine kinase from Thermococcus kodakarensis: cloning and characterization.

The genome sequence of Thermococcus kodakarensis contains an open reading frame, TK1110, annotated as ADP-dependent glucokinase. The encoding gene was expressed in Escherichia coli and the gene product, TK-GLK, was produced in soluble and active form. The recombinant enzyme was extremely thermostable. Thermostability was increased significantly in the presence of ammonium sulfate. ADP was the preferred co-factor for TK-GLK, which could be replaced with CDP but with a 60% activity. TK-GLK was a metal ion-dependent enzyme which exhibited glucokinase, glucosamine kinase and glucose 6-phosphatase activities. It catalyzed the phosphorylation of both glucose and glucosamine with nearly the same rate and affinity. The apparent Km values for glucose and glucosamine were 0.48 ± 0.03 and 0.47 ± 0.09 mM, respectively. The catalytic efficiency (kcat/Km) values against these two substrates were 6.2 × 105 ± 0.25 and 5.8 × 105 ± 0.75 M-1 s-1. The apparent Km value for dephosphorylation of glucose 6-phosphate was ~14-fold higher than that of glucose phosphorylation. Similarly, catalytic efficiency (kcat/Km) for phosphatase reaction was ~19-fold lower than that for the kinase reaction. To the best of our knowledge, this is the first report that describes the reversible nature of a euryarchaeal ADP-dependent glucokinase.

Effects of high-level expression of A1-ATPase on H2 production in Thermococcus kodakarensis.

The hyperthermophilic archaeon Thermococcus kodakarensis can grow on pyruvate or maltooligosaccharides through H2 fermentation. H2 production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H2 production is primary metabolism, here we aimed to partially uncouple cell growth and H2 production of T. kodakarensis. Additional A1-type ATPase genes were introduced into T. kodakarensis KU216 under the control of two promoters; the strong constitutive cell surface glycoprotein promoter, Pcsg, and the sugar-inducible fructose-1,6-bisphosphate aldolase promoter, Pfba. Whereas cells with the A1-type ATPase genes under the control of Pcsg displayed only trace levels of growth, cells with Pfba (strain KUA-PF) displayed growth sufficient for further analysis. Increased levels of A1-type ATPase protein were detected in KUA-PF cells grown on pyruvate or maltodextrin, when compared to the levels in the host strain KU216. The growth and H2 production levels of strain KUA-PF with pyruvate or maltodextrin as a carbon and electron source were analyzed and compared to those of the host strain KU216. Compared to a small decrease in total H2 production, significantly larger decreases in cell growth were observed, resulting in an increase in cell-specific H2 production. Quantification of the substrate also revealed that ATPase overexpression led to increased cell-specific pyruvate and maltodextrin consumptions. The results clearly indicate that ATPase production results in partial uncoupling of cell growth and H2 production in T. kodakarensis.

Affinity shift of ATP upon glycerol binding to a glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1.

Glycerol kinase (GK) is a key enzyme of glycerol metabolism. It participates in glycolysis and lipid membrane biosynthesis. A hexamer of GK from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1(Tk-GK) was identified as a substrate-binding form of the enzyme. Here, the X-ray crystal structure analysis and the biochemical analysis was done and the relationships between its unique oligomer structure and substrate binding affinity were investigated. Wild type GK and mutant K271E GK, which disrupts the hexamer formation interface, were crystallized with and without their substrates and analyzed at 2.19-3.05 Å resolution. In the absence of glycerol, Tk-GK was a dimer in solution. In the presence of its glycerol substrate, however, it became a hexamer consisting of three symmetrical dimers about the threefold axis. Through glycerol binding, all Tk-GK molecules in the hexamer were in closed form as a result of domain-motion. The closed form of Tk-GK had tenfold higher ATP affinity than the open form of Tk-GK. The hexamer structure stabilized the closed conformation and enhanced ATP binding affinity when the GK was bound to glycerol. This molecular mechanism is quite simple activity regulation mechanism among known GKs.

Heterologous gene expression and characterization of TK2246, a highly active and thermostable plant type l-asparaginase from Thermococcus kodakarensis.

The genome sequence of the hyperthermophilic archaeon Thermococcus kodakarensis contains two putative genes, TK1656 and TK2246, annotated as l-asparaginases. TK1656 has been reported previously. The current report is focused on TK2246, a plant-type l-asparaginase, which consists of 918 nucleotides corresponding to a polypeptide of 306 amino acids. The gene was cloned, expressed in Escherichia coli and the purified gene product was used to determine the properties of the recombinant enzyme. TK2246 was optimally active at 85 °C and pH 7.0 with a specific activity of 767 µmol min-1 mg-1 towards l-asparagine. The enzyme exhibited a 10% activity towards d-asparagine as compared to 100% against l-asparagine. No detectable activity was observed towards l- or d-glutamine. Half-life of the enzyme was nearly 18 h at 85 °C. TK2246 exhibited apparent Km and Vmax values of 3.1 mM and 833 µmol min-1 mg-1, respectively. Activation energy of the reaction, determined from the Arrhenius plot, was 28.3 kJ mol-1. To the best of our knowledge, this is the first characterization of a plant-type l-asparaginase from class Thermococci of phylum Euryarchaeota.

Branched-chain polyamine stabilizes RNA polymerase at elevated temperatures in hyperthermophiles.

Branched-chain polyamines (BCPAs) are unique polycations found in (hyper)thermophiles. Thermococcus kodakarensis grows optimally at 85 °C and produces the BCPA N4-bis(aminopropyl)spermidine by sequential addition of decarboxylated S-adenosylmethionine (dcSAM) aminopropyl groups to spermidine (SPD) by BCPA synthase A (BpsA). The T. kodakarensis bpsA deletion mutant (DBP1) did not grow at temperatures at or above 93 °C, and grew at 90 °C only after a long lag period following accumulation of excess cytoplasmic SPD. This suggests that BCPA plays an essential role in cell growth at higher temperatures and raises the possibility that BCPA is involved in controlling gene expression. To examine the effects of BCPA on transcription, the RNA polymerase (RNAP) core fraction was extracted from another bpsA deletion mutant, DBP4 (RNAPDBP4), which carried a His-tagged rpoL, and its enzymatic properties were compared with those of RNAP from wild-type (WT) cells (RNAPWT). LC-MS analysis revealed that nine ribosomal proteins were detected from RNAPWT but only one form RNAPDBP4. These results suggest that BCPA increases the linkage between RNAP and ribosomes to achieve efficient coupling of transcription and translation. Both RNAPs exhibited highest transcription activity in vitro at 80 °C, but the specific activity of RNAPDBP4 was lower than that of RNAPWT. Upon addition of SPD and BCPA, both increased the transcriptional activity of RNAPDBP4; however, elevation by BCPA was achieved at a tenfold lower concentration. Addition of BCPA also protected RNAPDBP4 against thermal inactivation at 90 °C. These results suggest that BCPA increases transcriptional activity in T. kodakarensis by stabilizing the RNAP complex at high temperatures.

Crystal structure of pantoate kinase from Thermococcus kodakarensis.

The coenzyme A biosynthesis pathways in most archaea involve two unique enzymes, pantoate kinase and phosphopantothenate synthetase, to convert pantoate to 4'-phosphopantothenate. Here, we report the first crystal structure of pantoate kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complex with ATP and a magnesium ion. The electron density for the adenosine moiety of ATP was very weak, which most likely relates to its broad nucleotide specificity. Based on the structure of the active site that contains a glycerol molecule, the pantoate binding site and the roles of the highly conserved residues are suggested.

Characterization of the copper-sensing transcriptional regulator CopR from the hyperthermophilic archeaon Thermococcus onnurineus NA1.

A putative copper ion-sensing transcriptional regulator CopR (TON_0836) from Thermococcus onnurineus NA1 was characterized. The CopR protein consists of a winged helix-turn-helix DNA-binding domain in the amino-terminal region and a TRASH domain that is assumed to be involved in metal ion-sensing in the carboxyl-terminal region. The CopR protein was most strongly bound to a region between its own gene promoter and a counter directional promoter region for copper efflux system CopA. When the divalent metals such as nickel, cobalt, copper, and iron were present, the CopR protein was dissociated from the target promoters on electrophoretic mobility shift assay (EMSA). The highest sensible ion is copper which affected protein releasing under 10 µM concentrations. CopR recognizes a significant upstream region of TATA box on CopR own promoter and acts as a transcriptional repressor in an in vitro transcription assay. Through site-directed mutagenesis of the DNA-binding domain, R34M mutant protein completely lost the DNA-binding activity on target promoter. When the conserved cysteine residues in C144XXC147 motif 1 of the TRASH domain were mutated into glycine, the double cysteine residue mutant protein alone lost the copper-binding activity. Therefore, CopR is a copper-sensing transcriptional regulator and acts as a repressor for autoregulation and for a putative copper efflux system CopA of T. onnurineus NA1.

Second Messenger cA4 Formation within the Composite Csm1 Palm Pocket of Type III-A CRISPR-Cas Csm Complex and Its Release Path.

Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cAn) formation from ATP subunits positioned within the composite pair of Palm domain pockets of the Csm1 subunit. The generated cAn second messenger in turn targets the CARF domain of trans-acting RNase Csm6, triggering its HEPN domain-based RNase activity. We have undertaken cryo-EM studies on multi-subunit Thermococcus onnurineus Csm effector ternary complexes, as well as X-ray studies on Csm1-Csm4 cassette, both bound to substrate (AMPPNP), intermediates (pppAn), and products (cAn), to decipher mechanistic aspects of cAn formation and release. A network of intermolecular hydrogen bond alignments accounts for the observed adenosine specificity, with ligand positioning dictating formation of linear pppAn intermediates and subsequent cAn formation by cyclization. We combine our structural results with published functional studies to highlight mechanistic insights into the role of the Csm effector complex in mediating the cAn signaling pathway.

CRISPR-Cas III-A Csm6 CARF Domain Is a Ring Nuclease Triggering Stepwise cA4 Cleavage with ApA>p Formation Terminating RNase Activity.

Type III-A CRISPR-Cas surveillance complexes containing multi-subunit Csm effector, guide, and target RNAs exhibit multiple activities, including formation of cyclic-oligoadenylates (cAn) from ATP and subsequent cAn-mediated cleavage of single-strand RNA (ssRNA) by the trans-acting Csm6 RNase. Our structure-function studies have focused on Thermococcus onnurineus Csm6 to deduce mechanistic insights into how cA4 binding to the Csm6 CARF domain triggers the RNase activity of the Csm6 HEPN domain and what factors contribute to regulation of RNA cleavage activity. We demonstrate that the Csm6 CARF domain is a ring nuclease, whereby bound cA4 is stepwise cleaved initially to ApApApA>p and subsequently to ApA>p in its CARF domain-binding pocket, with such cleavage bursts using a timer mechanism to regulate the RNase activity of the Csm6 HEPN domain. In addition, we establish T. onnurineus Csm6 as an adenosine-specific RNase and identify a histidine in the cA4 CARF-binding pocket involved in autoinhibitory regulation of RNase activity.