The effect of pseudoknot base pairing on cotranscriptional structural switching of the fluoride riboswitch.

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of Escherichia coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37°C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65°C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

A Novel Trehalose Synthase for the Production of Trehalose and Trehalulose.

A novel putative trehalose synthase gene (treM) was identified from an extreme temperature thermal spring. The gene was expressed in Escherichia coli followed by purification of the protein (TreM). TreM exhibited the pH optima of 7.0 for trehalose and trehalulose production, although it was functional and stable in the pH range of 5.0 to 8.0. Temperature activity profiling revealed that TreM can catalyze trehalose biosynthesis in a wide range of temperatures, from 5°C to 80°C. The optimum activity for trehalose and trehalulose biosynthesis was observed at 45°C and 50°C, respectively. A catalytic reaction performed at the low temperature of 5°C yielded trehalose with significantly reduced by-product (glucose) production in the reaction. TreM displayed remarkable thermal stability at optimum temperatures, with only about 20% loss in the activity after heat (50°C) exposure for 24 h. The maximum bioconversion yield of 74% trehalose (at 5°C) and 90% trehalulose (at 50°C) was obtained from 100 mM maltose and 70 mM sucrose, respectively. TreM was demonstrated to catalyze trehalulose biosynthesis utilizing the low-cost feedstock jaggery, cane molasses, muscovado, and table sugar. IMPORTANCE Trehalose is a rare sugar of high importance in biological research, with its property to stabilize cell membrane and proteins and protect the organism from drought. It is instrumental in the cryopreservation of human cells, e.g., sperm and blood stem cells. It is also very useful in the food industry, especially in the preparation of frozen food products. Trehalose synthase is a glycosyl hydrolase 13 (GH13) family enzyme that has been reported from about 22 bacterial species so far. Of these enzymes, to date, only two have been demonstrated to catalyze the biosynthesis of both trehalose and trehalulose. We have investigated the metagenomic data of an extreme temperature thermal spring to discover a novel gene that encodes a trehalose synthase (TreM) with higher stability and dual transglycosylation activities of trehalose and trehalulose biosynthesis. This enzyme is capable of catalyzing the transformation of maltose to trehalose and sucrose to trehalulose in a wide pH and temperature range. The present investigation endorses the thermal aquatic habitat as a promising genetic resource for the biocatalysts with high potential in producing high-value rare sugars.

Molecular basis for DarT ADP-ribosylation of a DNA base.

ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6-9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.

Role of bacterial RNA polymerase gate opening dynamics in DNA loading and antibiotics inhibition elucidated by quasi-Markov State Model.

To initiate transcription, the holoenzyme (RNA polymerase [RNAP] in complex with σ factor) loads the promoter DNA via the flexible loading gate created by the clamp and ß-lobe, yet their roles in DNA loading have not been characterized. We used a quasi-Markov State Model (qMSM) built from extensive molecular dynamics simulations to elucidate the dynamics of Thermus aquaticus holoenzyme's gate opening. We showed that during gate opening, ß-lobe oscillates four orders of magnitude faster than the clamp, whose opening depends on the Switch 2's structure. Myxopyronin, an antibiotic that binds to Switch 2, was shown to undergo a conformational selection mechanism to inhibit clamp opening. Importantly, we reveal a critical but undiscovered role of ß-lobe, whose opening is sufficient for DNA loading even when the clamp is partially closed. These findings open the opportunity for the development of antibiotics targeting ß-lobe of RNAP. Finally, we have shown that our qMSMs, which encode non-Markovian dynamics based on the generalized master equation formalism, hold great potential to be widely applied to study biomolecular dynamics.

Structural and functional studies of SF1B Pif1 from Thermus oshimai reveal dimerization-induced helicase inhibition.

Pif1 is an SF1B helicase that is evolutionarily conserved from bacteria to humans and plays multiple roles in maintaining genome stability in both nucleus and mitochondria. Though highly conserved, Pif1 family harbors a large mechanistic diversity. Here, we report crystal structures of Thermus oshimai Pif1 (ToPif1) alone and complexed with partial duplex or single-stranded DNA. In the apo state and in complex with a partial duplex DNA, ToPif1 is monomeric with its domain 2B/loop3 adopting a closed and an open conformation, respectively. When complexed with a single-stranded DNA, ToPif1 forms a stable dimer with domain 2B/loop3 shifting to a more open conformation. Single-molecule and biochemical assays show that domain 2B/loop3 switches repetitively between the closed and open conformations when a ToPif1 monomer unwinds DNA and, in contrast with other typical dimeric SF1A helicases, dimerization has an inhibitory effect on its helicase activity. This mechanism is not general for all Pif1 helicases but illustrates the diversity of regulation mechanisms among different helicases. It also raises the possibility that although dimerization results in activation for SF1A helicases, it may lead to inhibition for some of the other uncharacterized SF1B helicases, an interesting subject warranting further studies.

Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1.

Several native and engineered heat-stable DNA polymerases from a variety of sources are used as powerful tools in different molecular techniques, including polymerase chain reaction, medical diagnostics, DNA sequencing, biological diversity assessments, and in vitro mutagenesis. The DNA polymerase from the extreme thermophile, Thermus scotoductus strain K1, (TsK1) was expressed in Escherichia coli, purified, and characterized. This enzyme belongs to a distinct phylogenetic clade, different from the commonly used DNA polymerase I enzymes, including those from Thermus aquaticus and Thermus thermophilus. The enzyme demonstrated an optimal temperature and pH value of 72-74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K+ ions but it did need Mg2+ at 3-5 mM for optimal activity. It was stable for at least 1 h at 80°C, and its half-life at 88 and 95°C was 30 and 15 min, respectively. Analysis of the mutation frequency in the amplified products demonstrated that the base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase. These results suggest that TsK1 DNA polymerase could be useful in various molecular applications, including high-temperature DNA polymerization.

Comparison of Catalyzing Properties of Bacterial 4-α-Glucanotransferases Focusing on Their Cyclizing Activity.

A newly cloned 4-α-glucanotransferase (αGT) from Deinococcus geothermalis and two typical bacterial αGTs from Thermus scotoductus and Escherichia coli (MalQ) were investigated. Among 4 types of catalysis, the cyclization activity of αGTs that produces cycloamylose (CA), a valuable carbohydrate making inclusion complexes, was intensively studied. The new αGT, DgαGT, showed close protein sequence to the αGT from T. scotoductus (TsαGT). MalQ was clearly separated from the other two αGTs in the phylogenetic and the conserved regions analyses. The reaction velocities of disproportionation, cyclization, coupling, and hydrolysis of three αGTs were determined. Intriguingly, MalQ exhibited more than 100-fold lower cyclization activity than the others. To lesser extent, the disproportionation activity of MalQ was relatively low. DgαGT and TsαGT showed similar kinetics results, but TsαGT had nearly 10-fold lower hydrolysis activity than DgαGT. Due to the very low cyclizing activity of MalQ, DgαGT and TsαGT were selected for further analyses. When amylose was treated with DgαGT or TsαGT, CA with a broad DP range was generated immediately. The DP distribution of CA had a bimodal shape (DP 7 and 27 as peaks) for the both enzymes, but larger DPs of CA quickly decreased in the DgαGT. Cyclomaltopentaose, a rare cyclic sugar, was produced at early reaction stage and accumulated as the reactions went on in the both enzymes, but the increase was more profound in the TsαGT. Taken together, we clearly demonstrated the catalytic differences between αGT groups from thermophilic and pathogenic bacteria that and showed that αGTs play different roles depending on their lifestyle.

Structural evidence for a reaction intermediate mimic in the active site of a sulfite dehydrogenase.

By combining X-ray crystallography, electron paramagnetic resonance techniques and density functional theory-based modelling, we provide evidence for a direct coordination of the product analogue, phosphate, to the molybdenum active site of a sulfite dehydrogenase. This interaction is mimicking the still experimentally uncharacterized reaction intermediate proposed to arise during the catalytic cycle of this class of enzymes. This work opens new perspectives for further deciphering the reaction mechanism of this nearly ubiquitous class of oxidoreductases.

Distinct Expression of the Two NO-Forming Nitrite Reductases in Thermus antranikianii DSM 12462T Improved Environmental Adaptability.

Hot spring ecosystems are analogous to some thermal environments on the early Earth and represent ideal models to understand life forms and element cycling on the early Earth. Denitrification, an important component of biogeochemical nitrogen cycle, is highly active in hot springs. Nitrite (NO2-) reduction to nitric oxide (NO) is the significant and rate-limiting pathway in denitrification and is catalyzed by two types of nitrite reductases, encoded by nirS and nirK genes. NirS and NirK were originally considered incompatible in most denitrifying organisms, although a few strains have been reported to possess both genes. Herein, we report the functional division of nirS and nirK in Thermus, a thermophilic genus widespread in thermal ecosystems. Transcriptional levels of nirS and nirK coexisting in Thermus antranikianii DSM 12462T were measured to assess the effects of nitrite, oxygen, and stimulation time. Thirty-nine Thermus strains were used to analyze the phylogeny and distribution of nirS and nirK; six representative strains were used to assess the denitrification phenotype. The results showed that both genes were actively transcribed and expressed independently in T. antranikianii DSM 12462T. Strains with both nirS and nirK had a wider range of nitrite adaptation and revealed nir-related physiological adaptations in Thermus: nirK facilitated adaptation to rapid changes and extended the adaptation range of nitrite under oxygen-limited conditions, while nirS expression was higher under oxic and relatively stable conditions.

Amylomaltase from Thermus filiformis: expression in Saccharomyces cerevisiae and its use in starch modification.

AIM: To express amylomaltase from Thermus filiformis (TfAM) in a generally recognized as safe (GRAS) organism and to use the enzyme in starch modification. METHODS AND RESULTS: TfAM was expressed in Saccharomyces cerevisiae, using 2% (w/v) galactose inducer under GAL1 promoter. The enzyme was thermostable with high disproportionation and cyclization activities. The main large-ring cyclodextrin (CD) products were CD24-CD29, with CD26 as maximum at all incubation times. TfAM was used to modify cassava and pea starches, the amylose content decreased 18% and 30%, respectively, when 5% (w/v) starch was treated with 0·5 U TfAM g-1 starch. The increase in short branched chain (DP, degree of polymerization, 1-5) and the broader chain length distribution pattern which extended to the longer chain (DP40) after TfAM treatment were observed. The thermal property was changed, with an increase in retrogradation of starch as suggested by a lower enthalpy. CONCLUSIONS: TfAM was successfully expressed in S. cerevisiae and was used to make starches with new functionality. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the expression of AM in the GRAS yeast and the production of a modified starch gel from pea starch to improve the versatility of starch for food use.

Engineering novel S-glycosidase activity into extremo-adapted ß-glucosidase by rational design.

The breakdown of sulphur glycosidic bonds in thioglycosides can produce isothiocyanate, a chemoprotective agent linked to the prevention of cancers; however, only a handful of enzymes have been identified that are k0nown to catalyse this reaction. Structural studies of the myrosinase enzyme, which is capable of hydrolysing the thioglycosidic bond, have identified residues that may play important roles in sulphur bond specific activity. Using rational design, two extremo-adapted ß-glycosidases from the species Thermus nonproteolyticus (TnoGH1) and Halothermothrix orenii (HorGH1) were engineered towards thioglycoside substrates. Twelve variants, six for TnoGH1and six for HorGH1, were assayed for activity. Remarkable enhancement of the specificity (kcat/KM) of TnoGH1 and HorGH1 towards ß-thioglycoside was observed in the single mutants TnoGH1-V287R (2500 M-1 s-1) and HorGH1-M229R (13,260 M-1 s-1) which showed a 3-fold increase with no loss in turnover rate when compared with the wild-type enzymes. Thus, the role of arginine is key to induce ß-thioglycosidase activity. Thorough kinetic investigation of the different mutants has shed light on the mechanism of ß-glycosidases when acting on the native substrate.Key Points •Key residues were identified in the active site of Brevicoryne brassicae myrosinase. •Rationally designed mutations were introduced into two extremo-adapted ß-glycosidases. •ß-glycosidases mutants exhibited improved activity against thioglycosidic bonds. •The mutation to arginine in the active site yielded the best variant.

Structural basis for electron transport mechanism of complex I-like photosynthetic NAD(P)H dehydrogenase.

NAD(P)H dehydrogenase-like (NDH) complex NDH-1L of cyanobacteria plays a crucial role in cyclic electron flow (CEF) around photosystem I and respiration processes. NDH-1L couples the electron transport from ferredoxin (Fd) to plastoquinone (PQ) and proton pumping from cytoplasm to the lumen that drives the ATP production. NDH-1L-dependent CEF increases the ATP/NADPH ratio, and is therefore pivotal for oxygenic phototrophs to function under stress. Here we report two structures of NDH-1L from Thermosynechococcus elongatus BP-1, in complex with one Fd and an endogenous PQ, respectively. Our structures represent the complete model of cyanobacterial NDH-1L, revealing the binding manner of NDH-1L with Fd and PQ, as well as the structural elements crucial for proper functioning of the NDH-1L complex. Together, our data provides deep insights into the electron transport from Fd to PQ, and its coupling with proton translocation in NDH-1L.

The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases.

OLD family nucleases contain an N-terminal ATPase domain and a C-terminal Toprim domain. Homologs segregate into two classes based on primary sequence length and the presence/absence of a unique UvrD/PcrA/Rep-like helicase gene immediately downstream in the genome. Although we previously defined the catalytic machinery controlling Class 2 nuclease cleavage, degenerate conservation of the C-termini between classes precludes pinpointing the analogous residues in Class 1 enzymes by sequence alignment alone. Our Class 2 structures also provide no information on ATPase domain architecture and ATP hydrolysis. Here we present the full-length structure of the Class 1 OLD nuclease from Thermus scotoductus (Ts) at 2.20 Å resolution, which reveals a dimerization domain inserted into an N-terminal ABC ATPase fold and a C-terminal Toprim domain. Structural homology with genome maintenance proteins identifies conserved residues responsible for Ts OLD ATPase activity. Ts OLD lacks the C-terminal helical domain present in Class 2 OLD homologs yet preserves the spatial organization of the nuclease active site, arguing that OLD proteins use a conserved catalytic mechanism for DNA cleavage. We also demonstrate that mutants perturbing ATP hydrolysis or DNA cleavage in vitro impair P2 OLD-mediated killing of recBC-Escherichia coli hosts, indicating that both the ATPase and nuclease activities are required for OLD function in vivo.

Production and characterization of a recombinant thermophilic trehalose synthase from Thermus antranikianii.

Trehalose synthase converts maltose into trehalose in a single conversion step via intramolecular transformation and is thus useful for industrial production. In this study, we synthesized a thermophilic trehalose synthase from Thermus antranikianii (TaTS), which was recombinantly expressed in Escherichia coli BL21(DE3). The recombinant TaTS showed the highest activity at pH 7.0 and 60°C, with the maximum trehalose yield (76.8%) obtained at pH 7.0 and 30°C. TaTS activity was stable over a wide pH and temperature range of 6-10 and 4-70°C, respectively, over 6 h of incubation. The enzyme activity was strongly inhibited by Co2+, Cu2+, Zn2+, sodium dodecyl sulfate, and Tris. TaTS showed a 1.48-fold higher catalytic efficiency (kcat/Km) for maltose than for trehalose. Overall, these results demonstrate the good application potential of the recombinant enzyme TaTS in the efficient conversion of trehalose from maltose, with superior environmental tolerance to other trehalose synthases reported.

Thermostable Amylosucrase from Calidithermus timidus DSM 17022: Insight into Its Characteristics and Tetrameric Conformation.

Amylosucrase (EC 2.4.1.4, ASase), a typical carbohydrate-active enzyme, can catalyze 5 types of reactions and recognize more than 50 types of glycosyl acceptors. However, most ASases are unstable even at 50 °C, which limits their practical industrial applications. In this study, an extremely thermostable ASase was discovered from Calidithermus timidus DSM 17022 (CT-ASase) with an optimal activity temperature of 55 °C, half-life of 1.09 h at 70 °C, and melting temperature of 74.47 °C. The recombinant CT-ASase was characterized as the first tetrameric ASase, and a structure-based truncation mutation was conducted to confirm the effect of tetrameric conformation on its thermostability. In addition, α-1,4-glucan was found to be the predominant product of CT-ASase at pH 6.0-8.0 and 30-60 °C.

Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase.

We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl2 concentrations. The biallelic KO samples can be judged as 'negative' under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstream of a target gene's proto-spacer adjacent motif (PAM), and the reverse primer corresponds to the sequence ~200 bp downstream from the PAM. PCR performed using this primer set under standard MgCl2 concentrations (1.5-2.5 mM) should generate PCR products derived from both mutated and unedited alleles, whereas PCR performed using lower MgCl2 concentrations (0.8-2 mM) should yield products derived from unedited alleles. This enables high-throughput screening of biallelic mutants among cells/embryos having ≥1 indels at a region within 5 bp upstream of the PAM (where more than 94% of indels are known to appear). We performed proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp1, Ramp3, and Rosa26 mouse samples carrying various types of indels, and demonstrate that this new technique allows rapid identification of biallelic KO mutants among samples carrying various types of indels and mosaic mutations with 100% accuracy. We name this system detection of biallelic KO mutants harbouring indels using PCR (Bindel-PCR).

Impact of aqualysin 1 peptidase from Thermus aquaticus on molecular scale changes in the wheat gluten network during bread baking.

The impact of Aqualysin 1 (Aq1), the thermo-active peptidase of Thermus aquaticus, on wheat albumin, globulin, gliadin and glutenin proteins during heat treatment of wheat dough and bread baking was examined. The level of protein extractable in sodium dodecyl sulfate containing medium under non-reducing conditions (SDS-EP-NR) from wheat dough decreases upon heating to a lesser extent when Aq1 is used than in control experiments. The higher SDS-EP-NR level is caused by the release by Aq1 of peptides from the repetitive gluten protein domains during baking. These peptides are also extractable from bread crumb with salt solution. The resultant thermoset gluten network in bread crumb is mainly made up by protein from non-repetitive gluten domains.

Insights into the mode of flavin mononucleotide binding and catalytic mechanism of bacterial chromate reductases: A molecular dynamics simulation study.

Enzymes from natural sources protect the environment via complex biological mechanisms, which aid in reductive immobilization of toxic metals including chromium. Nevertheless, progress was being made in elucidating high-resolution crystal structures of reductases and their binding with flavin mononucleotide (FMN) to understand the underlying mechanism of chromate reduction. Therefore, herein, we employed molecular dynamics (MD) simulations, principal component analysis (PCA), and binding free energy calculations to understand the dynamics behavior of these enzymes with FMN. Six representative chromate reductases in monomeric and dimeric forms were selected to study the mode, dynamics, and energetic component that drive the FMN binding process. As evidenced by MD simulation, FMN prefers to bind the cervix formed between the catalytic domain surrounded by strong conserved hydrogen bonding, electrostatic, and hydrophobic contacts. The slight movement and reorientation of FMN resulted in breakage of some crucial H-bonds and other nonbonded contacts, which were well compensated with newly formed H-bonds, electrostatic, and hydrophobic interactions. The critical residues aiding in tight anchoring of FMN within dimer were found to be strongly conserved in the bacterial system. The molecular mechanics combined with the Poisson-Boltzmann surface area binding free energy of the monomer portrayed that the van der Waals and electrostatic energy contribute significantly to the total free energy, where, the polar solvation energy opposes the binding of FMN. The proposed proximity relationships between enzyme and FMN binding site presented in this study will open up better avenues to engineer enzymes with optimized chromate reductase activity for sustainable bioremediation of heavy metals.

A Molecular Dynamics Investigation of the Thermostability of Cold-Sensitive I707L KlenTaq1 DNA Polymerase and Its Wild-Type Counterpart.

DNA polymerase I from Thermus aquaticus ( Taq DNA polymerase) is useful for polymerase chain reactions because of its exceptional thermostability; however, its activity at low temperatures can cause amplification of unintended products. Mutation of isoleucine 707 to leucine (I707L) slows Taq DNA polymerase at low temperatures, which decreases unwanted amplification due to mispriming. In this work, unrestrained molecular dynamics (MD) simulations were performed on I707L and wild-type (WT) Taq DNA polymerase at 341 and 298 K to determine how the mutation affects the dynamic nature of the protein. The results suggest that I707L Taq DNA polymerase remains relatively immobile at room temperature and becomes more flexible at the higher temperature, while the WT Taq DNA polymerase demonstrates less substantial differences in dynamics at high and low temperatures. These results are in agreement with previous experimental results on the I707L mutant Taq DNA polymerase that show dynamic differences at high and low temperatures. The decreased mobility of the mutant at low temperature suggests that the mutant remains longer in the blocked conformation, and this may lead to reduced activity relative to the WT at 298 K. Principal component analysis revealed that the mutation results in decoupled movements of the Q helix and fingers domain. This decoupled nature of the mutant gives way to an increasingly flexible N-terminal end of the Q helix at 341 K, a characteristic not seen for WT Taq DNA polymerase.

Novel parameter describing restriction endonucleases: Secondary-Cognate-Specificity and chemical stimulation of TsoI leading to substrate specificity change.

Over 470 prototype Type II restriction endonucleases (REases) are currently known. Most recognise specific DNA sequences 4-8 bp long, with very few exceptions cleaving DNA more frequently. TsoI is a thermostable Type IIC enzyme that recognises the DNA sequence TARCCA (R = A or G) and cleaves downstream at N11/N9. The enzyme exhibits extensive top-strand nicking of the supercoiled single-site DNA substrate. The second DNA strand of such substrate is specifically cleaved only in the presence of duplex oligonucleotides containing a cognate site. We have previously shown that some Type IIC/IIG/IIS enzymes from the Thermus-family exhibit 'affinity star' activity, which can be induced by the S-adenosyl-L-methionine (SAM) cofactor analogue-sinefungin (SIN). Here, we define a novel type of inherently built-in 'star' activity, exemplified by TsoI. The TsoI 'star' activity cannot be described under the definition of the classic 'star' activity as it is independent of the reaction conditions used and cannot be separated from the cognate specificity. Therefore, we define this phenomenon as Secondary-Cognate-Specificity (SCS). The TsoI SCS comprises several degenerated variants of the cognate site. Although the efficiency of TsoI SCS cleavage is lower in comparison to the cognate TsoI recognition sequence, it can be stimulated by S-adenosyl-L-cysteine (SAC). We present a new route for the chemical synthesis of SAC. The TsoI/SAC REase may serve as a novel tool for DNA manipulation.