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We present a new class of DNA-based nanoswitches that, upon enzymatic repair, could undergo a conformational change mechanism leading to a change in fluorescent signal. Such folding-upon-repair DNA nanoswitches are synthetic DNA sequences containing O(6)-methyl-guanine (O(6)-MeG) nucleobases and labelled with a fluorophore/quencher optical pair. The nanoswitches are rationally designed so that only upon enzymatic demethylation of the O(6)-MeG nucleobases they can form stable intramolecular Hoogsteen interactions and fold into an optically active triplex DNA structure. We have first characterized the folding mechanism induced by the enzymatic repair activity through fluorescent experiments and Molecular Dynamics simulations. We then demonstrated that the folding-upon-repair DNA nanoswitches are suitable and specific substrates for different methyltransferase enzymes including the human homologue (hMGMT) and they allow the screening of novel potential methyltransferase inhibitors.

In the past decade, a powerful biotechnological tool for specific in vivo and in vitro labeling of proteins of interest, the SNAP-tag technology, has been proposed as a valid alternative to the classic protein-tags. This self-labeling protein-tag is an engineered variant of the human O6-alkylguanine-DNA-alkyltransferases (hMGMT) enzyme, which specifically reacts with benzyl-guanine (BG) derivatives, maintaining a part of these substrates in the catalytic site, with a one-step reaction. However, given the mesophilic nature of this protein-tag, the general use of this new approach is restricted to mesophilic model systems and mild reaction conditions. To overcome this limitation, it is necessary the exploitation of homologous activities from extremophilic sources, to apply this technology also to (hyper)thermophilic organisms and harsher reaction conditions. To this purpose, it has been first characterized an archaeal alkylguanine-DNA-alkyltransferases (AGT or OGT) from Saccharolobus solfataricus (SsOGT), which presented an exceptional in vitro stability at high temperatures, physical and chemical denaturing agents, making it suitable not only for the in vivo heterologous expression in Escherichia coli, but also in the thermophilic Thermus thermophilus and Sulfolobus islandicus organisms. Furthermore, the growing demand to apply this biotechnological tool to extreme conditions led to look for new protein-tags from hot sources. Recently, new AGTs from Thermotoga neapolitana (TnOGT) and Pyrococcus furiosus (PfuOGT) resulted different from SsOGT in terms of activity and stability, proposing them as potential tool for the in vivo analysis and function of enzymes of interest in these hyperthermophilic model systems.

Alkylguanine-DNA-alkyltransferases (AGTs) have a natural role in the protection of DNA from mutations caused by alkylating agents. Their peculiar irreversible self-alkylation reaction led to the development as new tools in the modern biotechnology. SNAP-tag® is a powerful enzyme for the specific labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. This technology has some limitations, as the mesophilic nature of the tag (an engineered variant of the human enzyme) and the needs of purify each substrate. Recently, the SNAP-tag® technology was successfully implemented by the employment of thermostable “SNAP-tag-like” variants from (hyper)thermophilic sources, and by the utilization of BG-substrates containing an azide group to be combined with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. The introduction of these new actors on the scene made possible the expansion of the methodology to in vivo and in vitro harsh reaction conditions, as well as the utilization of more chemical groups in the overall reaction enzyme labelling.

The use of natural systems, such as outer membrane protein A (OmpA), phosphoporin E (PhoE), ice nucleation protein (INP), etc., has been proved very useful for the surface exposure of proteins on the outer membrane of Gram-negative bacteria. These strategies have the clear advantage of unifying in a one-step the production, the purification and the in vivo immobilisation of proteins/biocatalysts onto a specific biological support. Here, we introduce the novel Anchoring-and-Self-Labelling-protein-tag (ASLtag), which allows the in vivo immobilisation of enzymes on E. coli surface and the labelling of the neosynthesised proteins with the engineered alkylguanine-DNA-alkyl-transferase (H5) from Sulfolobus solfataricus. Our results demonstrated that this tag enhanced the overexpression of thermostable enzymes, such as the carbonic anhydrase (SspCA) from Sulfurihydrogenibium yellowstonense and the β-glycoside hydrolase (SsβGly) from S. solfataricus, without affecting their folding and catalytic activity, proposing a new tool for the improvement in the utilisation of biocatalysts of biotechnological interest.

SNAP-tag® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a chemo-enzymatic approach, by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP-tag® and its relative thermostable version (SsOGT-H5) proved to be very active on this substrate. The stability of these tags upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling protein-tag.

The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as protein tags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications.

The specific labelling of proteins in recent years has made use of self-labelling proteins, such as the SNAP-tag(®) and the Halotag(®). These enzymes, by their nature or suitably engineered, have the ability to specifically react with their respective substrates, but covalently retaining a part of them in the catalytic site upon reaction. This led to the synthesis of substrates conjugated with, e.g., fluorophores (proposing them as alternatives to fluorescent proteins), but also with others chemical groups, for numerous biotechnological applications. Recently, a mutant of the OGT from Saccharolobus solfataricus (H(5)) very stable to high temperatures and in the presence of physical and chemical denaturing agents has been proposed as a thermostable SNAP-tag(®) for in vivo and in vitro harsh reaction conditions. Here, we show two new thermostable OGTs from Thermotoga neapolitana and Pyrococcus furiosus, which, respectively, display a higher catalytic activity and thermostability respect to H(5), proposing them as alternatives for in vivo studies in these extreme model organisms.

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

Background The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-d-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-d-xylans to remove successive d-xylose residues from the non-reducing termini. Results We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation. Conclusion Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions.