COMENZE is an EU-funded HORIZON MSCA Doctoral Network, driving innovation in the bio-based polymer industry through advanced enzyme engineering. By developing cutting-edge enzymatic strategies, we aim to enhance sustainable polymer design, improve end-of-life degradability, and enable chemical recyclability.

Through the training of ten Doctoral Candidates in computational and experimental enzyme engineering, COMENZE contributes to a greener, circular economy in line with the European Green Deal.

Learn more on project website

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“Lytic polysaccharide monooxygenases (LPMO) and cytochrome P450 (CYP) are copper- and iron-dependent, respectively, enzymatic systems that perform regio- and stereospecific oxidation of non-activated hydrocarbons in Nature. To control such reactions in modern industry and biotechnology is of utmost importance in creating products of value such as secondgeneration bioethanol and products of value for i.e. the pharmaceutical industry. Due to the major drawbacks of using CYPs, including their partially membrane bound nature and the requirement of a reductase in combination with reducing agents such as NAD(P)H to transfer electrons to the active site for oxygen activation, it is highly desirable to develop new type of catalyst that can perform the same type of reactions. An attractive alternative strategy is to engineer LPMOs to perform CYP catalysis. LPMOs are small, robust, easy to produce in large scale, and rigid water-soluble proteins with a plethora of electron donors. The extended, flat LPMO surface, with huge natural sequence variation and thus, likely, mutability, provides a fantastic scaffold for engineering access to the active site as well as substrate affinity. We propose to use LPMOs engineered to accommodate typical CYP substrates and immobilize this on solid supports to provide confinement necessary in bringing the oxygen species together with the C-H bond to be oxidized in a tailored, “”closed”” environment. Moreover, the rate of LPMO catalysis can be greatly enhanced compared to traditional CYP catalysis by the addition H2O2 in the presence of low, priming concentrations of an external reductant to achieve efficiency constants (kcat/Km) in the order of 106 M-1s-1, which is typical for peroxygenases. The proposed ground-breaking research fits excellently well with the work program “”Future and Emerging Technologies”” where the goal is to challenge current thinking.”

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Detection of the different phases of Mycobacterium tuberculosis infection and prediction of the development of tuberculosis disease progression by application of novel interferon gamma release assay

Tuberculosis (TB) has a high mortality rate due to the ability of the bacteria Mycobacterium tuberculosis (Mtb) to hide after infection and the lack of accurate and sensitive tests to detect different TB forms and disease progression. The EU-funded DET-TB project aims to determine and investigate new antigens that could be used for the detection of Mtb infection phases and as an accurate prognostic tool. The main goal is to better understand the molecular mechanisms of TB infection and identify the new antigens for the design of novel interferon-gamma release assay tests for the detection of different phases of infection and disease progression.

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Treatment of chronic wounds, often occurring in groups of patients suffering from circulatory insufficiency, diabetes or being immobilized for long periods of time is exceedingly problematic for modern medicine. Despite using many surgical procedures, including hydrosurgery, the latest hydrogel dressings and artificial skin, the wound treatment process is very complicated, expensive and it does not always lead to complete cure of the patient. Therefore, researchers are searching for natural bioactive compounds, which could be used as alternative methods of wound treatment or as a supporting therapy, when traditional methods do not give satisfactory results.

One of the methods, known for a long time, is maggot therapy with use of e.g., fly maggots. It gives promising results, even for patients with chronic wounds. Maggots positively affect the wound healing process by: removal of dead tissue from the wound, eliminating bacteria and stimulating the creation of new blood vessels (angiogenesis).

Despite the positive effect on the wound healing process, the application of living postembryonic insect forms is limited, due to the fact that most patients feel uncomfortable with such therapy. Therefore, the isolation of useful compounds from maggot secretions that can be used to replace the living maggots’ effect in wound healing process is a topic of ongoing studies. Recently, several compounds that stimulate the wound healing process have been isolated and described, mainly biologically active enzymes, proteins, and fatty acids. However, little attention has been paid to peptides and low molecular weight compounds present in secretion, which also exhibit antibacterial and pro-angiogenic activity.

The main goals of the proposed project are to identify peptides and low molecular weight compounds, their analogues and metabolites present in fly maggot secretions before and after larvae immunization, and also to determine their biological activity. Here we would like to evaluate antibacterial and pro-angiogenic activity of compounds identified in maggot secretions.

The proposed research will extend the knowledge on natural and biologically active compounds beneficial for chronic wound treatment. In addition, they will be the basis for the development of innovative treatment methods and complementary therapies that would improve the patient’s comfort during therapy and shorten its duration.

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Project (2021/43/D/NZ7/03119) is financed by :

Since the dawn of time, humanity encountered a great problem with agricultural pests – viruses, bacteria, undesirable fungi, plants, and animals. The largest group of pests are insects, affecting not only agriculture but also forest areas. Insects can be vectors of dangerous diseases and negatively affect the health of plants, animals, and humans. On the other hand, they are an extremely important part of the ecosystem. For example, approximately 75% of crop species in agricultural and horticultural cropping systems are pollinated by insects. Therefore, pesticides and insecticides has to be designed with extreme care.

Extensive research on insect physiology has provided a lot of information about their  anatomy, organs, structures, and functioning. Great amount of work were devoted to analysing the insects development process. It was found, that the juvenile hormone (JH) is crucial in the insects’ metamorphosis. Nowadays, it is thought, that the juvenile hormone epoxide hydrolase is a key enzyme in this process. The inhibition of JHEH could prevent the development of an adult form of insects and consequently lead to the degradation of particular species. It would be desirable in the case of insects that are agricultural and forest pests, but also in relation to insects, which can be vectors of dangerous diseases.

The proposed project brings together different fields of research: chemistry, biology, microbiology, biotechnology, entomology and ecology. In the proposed project, we would like to use in silico methods to identify differences in the structural properties and dynamics of JHEH among different insect of closely related species. By carrying out a comprehensive structural analysis focused mainly on the definition and description of intramolecular voids that are available for the binding of small molecules, it will be possible to identify regions in JHEH that are unique to individual insect species and potentially able to distinguish selective inhibitors dedicated to specific pests and safe to other insects, plants and animals. The proposed compounds will be tested also for environmental safety.

Project is financed by:

Weronika Bagrowska, Angelika Karasewicz, Artur Góra, Comprehensive analysis of acetylcholinesterase inhibitor and reactivator complexes: implications for drug design and antidote development. Drug Discovery Today 2024, 29(12), 104217, doi: 10.1016/j.drudis.2024.104217 ARTICLE

This project aims to identify, characterize and demonstrate the antimicrobial activity of bacteriocins derived from the marine environment. There is an increasing concern about antibiotics crisis in recent years due to the unconscious and extensive use of antibiotics, which causes resistant bacteria to evolve and decreases the effectiveness of the current selection of antibiotics available. In this project, we aim to explore and exploit the potential of Turkish marine habitats in terms of antimicrobials that can be used as an environment-friendly and what is the most important efficient alternative to antibiotics. The proposed project aims to identify bacterial peptides with antimicrobial activity, called bacteriocins, from the marine environment, characterize selected bacteriocins, test their antimicrobial activity against various human pathogens, investigate the synergistic activity of bacteriocins with antibiotics and determine bacteriocins’ environmental impacts falls within the scope of the call. Previous studies were mainly focused on bacteriocins produced by terrestrial species. Even though there are marine bacteriocin studies conducted, they usually did not provide an in-depth characterization. They are however very important and promising in the medical treatment, since they can persist in highly saline habitats. This study aims to benefit from a unique source, Sea of Marmara, which is subjected to high levels of anthropogenic pressures due to its proximity to the most populated city of Europe, Istanbul and small exchange of water with surrounding basins. Both sediment and water samples will be collected from Istanbul shores, Izmit Bay and Kocaeli Municipal Wastewater Treatment Plant, which were found to be rich in multiple drug-resistant bacteria. The expected outcomes of this project will be obtaining antibiotic alternatives from marine environments with complex microbial community and impacted by human activity, in-depth characterization of isolated bacteriocins’ innate and synergistic activity against human pathogens and revealing their potential to be used as a treatment/supplement that is safer for the environment. The main research activities will be (i) identification and isolation of bacteriocin genes (ii) expression and characterization of selected bacteriocins (iii) testing microbial activity of bacteriocins against human pathogens (iv) modeling and insight into structural characterization of most potent bacteriocins and (v) evaluation of the risk of the off-target activity.

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Plastics formed by synthetic polymers are durable materials that possess many desirable features, but their high resistance to biodegradability, once considered an advantage, is now one of the main causes of environmental problems in the world. Every year, due to inadequate recycling of plastics, millions of tons of waste accumulate in terrestrial and aquatic environments, exerting a damaging effect on them. The micro and nanoparticles of plastics, formed as a result of physical erosion processes, pose a new and previously unknown threat to human and animal health. Effective methods of degradation of these synthetic polymers are needed. An alternative and environmentally friendly method of degradation of plastics is enzymatic biocatalysis. It is known that some microorganisms can colonize the surface of plastics and are able to slowly degrade it, however, this applies only to selected plastics, and the enzymes and mechanisms of the process of hydrolysis and/or oxidation of synthetic polymers are not well understood.

The aim of the proposed project is to find and learn about enzymes capable of binding short oligomers of synthetic polymers. Synthetic polymers, that were selected as subjects of this research are commonly used plastics: polyurethane (PUR), polystyrene (PS) and polyethylene (PE). The gained knowledge will be used to redesign the selected enzymes to increase its stability, affinity and activity, and if it is possible to expand its substrate selectivity.

Modern in silico techniques will be applied in the project, which will include molecular dynamics simulations, molecular docking, protein-ligand interaction analysis and computational enzyme design. It is assumed that as a result of the project, using in silico techniques, it will be possible to learn about the molecular aspects responsible for recognizing and binding polymer chains. Identification of key amino acids in this process will allow to optimize the surface of the protein responsible for recognizing
the substrate in order to increase the activity and modify the substrate specificity of enzymes capable of degrading synthetic polymers. The results of the computational work will also be experimentally verified.

Project is financed by:

1. Katarzyna Szleper, Mateusz Cebula, Oksana Kovalenko, Artur Góra, Agata Raczyńska, PUR-GEN: A Web Server for Automated Generation of Polyurethane Fragment Libraries. Computational and Structural Biotechnology Journal, 2024 doi: 10.1016/j.csbj.2024.12.004 ARTICLE^{Open Access}
2. Agata Raczyńska, Artur Góra, Isabelle André. An overview on polyurethane degrading enzymes. Biotechnology Advances, 2024, 77, 108439, doi: 10.1016/j.biotechadv.2024.108439 ARTICLE

To this date, we performed over 2µs of classical molecular dynamics simulations (cMD) of both SARS and SARS-CoV-2 main proteases (Mpros) as well as nearly 1 µs of mixed-solvents molecular dynamics simulations (MixMD) with various cosolvents including acetonitrile, benzene, dimethylsulfoxide, methanol, phenol, and urea [1]. The combined small molecules’ tracking approach and local distribution analysis were used to analyse the conformational changes in the binding site, as well as to detect other potential binding sites that could be used for inhibitors design. Our results indicate, that there are large differences between plasticity and size of the SARS-CoV and SARS-CoV-2 Mpro protease and moreover, the access to the active site can be regulated by the C44-P52 loop which is carrying one of the unique for SARS-CoV-2 Mpro amino acid [1].

Additionally, the preliminary analysis of the potential mutability of the active site surrounding was performed. The single nucleotide substitutions were introduced to the SARS-CoV-2 main protease gene and their energetic contributions to protein stability were calculated. The most important message comes from the analysis of the potential mutability of the C44-P52 loop. The mutation of four of them has a stabilising effect for the protein and for the rest the effect is near-neutral. These results indicate that the future evolution of the Mpro protein in this region is likely to occur and can significantly reduce the potential use of the drugs developed at this stage of research due to a highly probable development of drug resistance of this virus through mutations. Therefore our study is of significant importance and can provide a contribution to the development of future therapeutic strategies.

The preliminary results. (A) Comparison of cosolvent distribution in the active site of SARS-CoV-2 and SARS-CoV Mpro proteins during MixMD simulations. (B) Predicted stability of the residues of the active site cavity. The green colour indicate residues prone to mutate.

1. Papaj K., et al. Evaluation of Xa inhibitors as potential inhibitors of the SARS-CoV-2 Mpro protease. PloS ONE 2022, 17 (1), e0262482 doi: 10.1371/journal.pone.0262482 ARTICLE^{Open Access}
2. Papaj K., et al. Investigation of Thiocarbamates as Potential Inhibitors of the SARS-CoV-2 Mpro Pharmaceuticals 2021, 14(11), 1153 doi: 10.3390/ph14111153 ARTICLE^{Open Access}
3. Fischer A., et al. Computational Selectivity Assessment of Protease Inhibitors against SARS-CoV-2 Int. J. Mol. Sci. 2021, 22(4), 2065 doi: 10.3390/ ijms22042065 ARTICLE^{Open Access}
4. Bzówka M., et al. Structural and Evolutionary Analysis Indicate That the SARS-CoV-2 Mpro Is a Challenging Target for Small-Molecule Inhibitor Design, Int. J. Mol. Sci. 2020, 21(7), 3099 doi: 10.3390/ijms21093099 ARTICLE^{Open Access}

Water molecules take part in metabolic reactions, they constitute an intracellular transport environment but also are involved in dynamic changes in protein structures, including transmembrane proteins. It is assumed that they can affect signal recognition, as well as conformational changes caused by interaction with ligands or adapter proteins, or the process of signal transmission across the cell membrane. 

In most of the known studies, the analysis of water behaviour in intermolecular interactions has been omitted. Therefore, this project aims to determine whether water molecules can actually act as a mediator in protein regulation. The biological system which fits well for this study is the family of transmembrane Toll-like receptors (TLRs). These receptors recognise and interact with a wide spectrum of compounds – from low molecular compounds, through nucleic acids, peptides, to whole proteins. TLRs are a class of proteins that play a key role in the innate immune system. 

The proper proteins regulation is crucial for the organisms’ homeostasis. It is important to know the greatest number of factors affecting the protein regulation and determining the signal transduction pathway in the case of transmembrane proteins. Studying the role that water molecules play in these processes can lead to a better understanding of the basics of protein regulation at the molecular level.
This is especially important for Toll-like receptors. Any abnormality in the regulation of these proteins can cause serious diseases. Examination of the molecular aspects of the regulation of Toll-like receptors along with the characteristics of intermolecular interactions is, therefore, a key point that will help to understand their functioning.

Bzówka M’; Bagrowska W.; Góra A.; Recent Advances in Studying Toll-like Receptors with the Use of Computational Methods. J. Chem. Inf. Model., 2023, 63, 12, 3669–3687 doi: 10.1021/acs.jcim.3c00419  ARTICLE^{Open Access}

Project is financed by:

grant no: 0141/DIA/2019/48

The genetic code is a set of rules used to translate the information encoded within the genetic material (DNA or RNA) into proteins. It is highly similar among all organisms. The code defines how codons specify particular amino acids which will be added consecutive during protein synthesis. A three-nucleotide codon in a nucleic acid sequence specifies a particular amino acid. Single nucleotide substitution (SNP) is a substitution of a single nucleotide at a specific position in the genome. SNPs might cause very subtle (i.e. silent or missense mutations) differences in the produced protein, while sometimes they result in a truncated, incomplete, and usually nonfunctional protein (i.e., nonsense mutation) by introducing a premature stop-codon into the transcribed mRNA.

Such substitutions might be the cause of several genetic disorders. Therefore, the RARE-ILLN project is run in close cooperation with the Maria Skłodowska-Curie National Research Institute of Oncology to ensure careful and detailed analysis. Using in silico methods, such as homology modeling, molecular dynamics simulations, and small molecules tracking approach, we are able to determine the structure of the protein of interests in which the substitution was find, and analyse the differences between the wild-type (protein without introduced substitution) and protein with introduced substitution that could be related with differences in proteins stability and functionality.

1. Mitusińska K., et al. Structural analysis of the effect of Asn107Ser mutation on Alg13 activity and Alg13-Alg14 complex formation and expanding the phenotypic variability of ALG13-CDG. Biomolecules 2022, 12(3), 398 doi: 10.3390/biom12030398
2. Jezela-Stanek A., et al. Proteins Structure Models in the Evaluation of Novel Variant (C.472_477del) in the MOSC2 Gene. Diagnostics 2020, 10, 821 doi:10.3390/diagnostics10100821

The WAT-PROBE project aims to investigate the concept of protein structure characterisation by the intramolecular voids inspection approach. This conceptually straightforward idea can provide an alternative, easy method for protein structure-function relationship analysis. We are using water molecules as molecular probes for protein structure interior examination. With the use of a ligand tracking and local density analysis approach, we aim to confirm the usability of such an analysis for: hot-spot identification, cavity and tunnel description, and investigation of the dynamics of the protein interior. In parallel, we wish to improve the existing methods which can be applied to such analysis, and we are going to validate water models used in molecular dynamics simulations for protein interior investigation. The basic knowledge obtained in the project will be used to study the role of water molecules penetrating enzymes with regard to their selectivity and activity. The results of the in silico study will be validated with experimental techniques. 

The analysis of transport pathways and cavities with water as a molecular probe can provide useful information for protein engineering. The proper analysis of water ‘behaviour’ inside the protein core can significantly improve the description of intrinsic transport pathways with access to both geometrical and physico-chemical information including the energy profile, and the detection of hot-spots for protein redesign. The second applicability of the project is closely related to drug design and enzyme specificity optimisation. By mapping the dynamics of cavities, we wish to explore the potential of enzyme adaptation to different substrates/ligands. In this part of the project we are including calculations with organic co-solvents to get access to a description of non-bonding interactions. The validation will be validated in two aspects, the application of the method in protein reengineering, and pharmacophore description.

The expected outcomes of proposed project are:

• further development of methods for small molecule tracking,
• validation of the usability of water molecules as a molecular probe for the study of protein dynamics,
• delivery of an alternative method with potential applicability in protein engineering and drug design,
• description of the role of the water inside the protein core of enzymes. 

The results of hot-spot detection (A), exploration of cavities by water molecules (B), and identified water leakage (blue pathway) (C) obtained with a recent version of the AQUA-DUCT software [1] during Solanum tuberosum epoxide hydrolase study. Please note that besides gates (indicated by green and orange balls on picture A), other functionally important residues were also detected by AQUA-DUCT software, (Figures from [2]).

1. Bzówka M. et al. Evolution of tunnels in α/β-hydrolase fold proteins—What can we learn from studying epoxide hydrolases? PLOS Computational Biology 2022, 18 (5), e1010119 doi: 10.1371/journal.pcbi.1010119  ARTICLE^{Open Access}
2. Mitusińska K. et al. Geometry-Based versus Small-Molecule Tracking Method for Tunnel Identification: Benefits and Pitfalls. J. Chem. Inf. Model. 2022, 62, 24, 6803–6811. doi: 10.1021/acs.jcim.2c00985 ARTICLE^{Open Access}
3. Mitusińka K. et al. Structure-function relationship between soluble epoxide hydrolases structure and their tunnel network. Computational and Structural Biotechnology Journal 2022, 20, 193-205. doi: 10.1016/j.csbj.2021.10.042 ARTICLE^{Open Access}
4. Mitusińska K., et al. AQUA-DUCT: Analysis of Molecular Dynamics Simulations of Macromolecules with the use of Molecular Probes [Article v1. 0]. Living Journal of Computational Molecular Science 2021, 2 (1), 21383 doi: 10.33011/livecoms.2.1.21383  ARTICLE^{Open Access}
5. Magdziarz, T., et al. AQUA-DUCT 1.0: structural and functional analysis of macromolecules from an intramolecular voids perspective. Bioinformatics 2020, 36(8), 2599-2601, doi:10.1093/bioinformatics/btz946 ARTICLE^{Open Access}
6. Mitusińska K., et al. Applications of water molecules for analysis of macromolecule properties. Computational and Structural Biotechnology Journal 2020, 18, 355-365, doi:10.1016/j.csbj.2020.02.001 ARTICLE^{Open Access}
7. Mitusińska, K., et al. Exploring Solanum tuberosum Epoxide Hydrolase Internal Architecture by Water Molecules Tracking. Biomolecules 2018, 8, 143, doi:10.3390/biom8040143 ARTICLE^{Open Access}
8. Magdziarz, T., et al. AQUA DUCT: A ligands tracking tool. Bioinformatics 2017, 33, 2045–2046, doi:10.1093/bioinformatics/btx125 ARTICLE

Software dedicated page:

Research on enzymes conducted during the last decade provided evidences that regions located further from the active sites can also determine enzymes properties. Such control may come from co-reagents or inhibitors providing allosteric regulations or may appear as a result of localisation of active site deep into protein core. In second case transport occurs through tunnels network. Substrate access pathways provide additional constrains for binding of ligands to the active site. Precise control may be achieved by gates – dynamic systems made of individual amino acids residues, loops, secondary structure elements or domains, which are able to change a geometrical state between open and closed conformation reversibly and by such transition controls the flow of small molecules – substrates, products, ions and solvents – in and out of the protein structure.

The gates in enzymes functionally may:

1. contribute to enzyme selectivity by controlling the access of substrates,
2. protect a specific region of a protein against a solvent access or
3. synchronize processes occurring in distinct parts of a protein.

The passage of molecules through the access pathways can be controlled by gates trough their specific molecular interactions, e.g., electrostatic, hydrophobic or their geometrical properties, e.g. size discrimination in the bottleneck. The proper function of the gates, even the simplest ones, may be indispensable for catalysis and the gating event can even represent the rate-limiting step of the catalytic cycle.

The expected outcomes of proposed project are:

1. detailed description of the role of gating and anchoring residues in studied systems,
2. identification of correlations between gating and anchoring residues,
3. verification of hypothesis about higher rate of evolution of gating residues comparing with entire protein sequence,
4. exploring the role of gating and anchoring residues in substrate/products transportation from/to active site of the enzyme,
5. establishment of methodology for fast and accurate identification of gating and anchoring residues.

Publications:

1. Bzówka M., et al. Evolution of tunnels in α/β-hydrolase fold proteins—What can we learn from studying epoxide hydrolases? PLOS Computational Biology 2022, 18 (5), e1010119 doi: 10.1371/journal.pcbi.1010119 ARTICLE^{Open Access}
2. Mitusińka K. et al. Structure-function relationship between soluble epoxide hydrolases structure and their tunnel network. Computational and Structural Biotechnology Journal 2022, 20, 193-205. doi: 10.1016/j.csbj.2021.10.042 ARTICLE^{Open Access}
3. Mitusińska K., et al. AQUA-DUCT: Analysis of Molecular Dynamics Simulations of Macromolecules with the use of Molecular Probes [Article v1. 0]. Living Journal of Computational Molecular Science 2021, 2 (1), 21383 doi: 10.33011/livecoms.2.1.21383
4. Mitusińska, K., et al. Applications of water molecules for analysis of macromolecule properties. Computational and Structural Biotechnology Journal 18, 355–365 (2020).
5. Magdziarz, T. et al. AQUA-DUCT 1.0: structural and functional analysis of macromolecules from an intramolecular voids perspective. Bioinformatics (2019). doi:10.1093/bioinformatics/btz946
6. Subramanian, K. et al. Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcus furiosus Phosphoglucose Isomerase. Biomolecules 9, 212 (2019)
7. Mitusińska, K. et al. Exploring Solanum tuberosum Epoxide Hydrolase Internal Architecture by Water Molecules Tracking. Biomolecules 8, 143 (2018)
8. Płuciennik, A. et al. BALCONY: an R package for MSA and functional compartments of protein variability analysis. BMC Bioinformatics 19, 300 (2018)
9. Subramanian, K. et al. Modulating D-amino acid oxidase (DAAO) substrate specificity through facilitated solvent access. PLoS One 13, e0198990 (2018)
10. Magdziarz, T. et al. AQUA-DUCT: A ligands tracking tool. Bioinformatics 33, 2045–2046 (2017)

Project is financed by:

grant no. UMO-2013/10/E/NZ1/00649

Project Partner – Loschmidt Laboratory, Brno, Czech Republic.

Enzymes are versatile proteins which catalyse most of reactions within the body. The structure of enzymes was optimised to perform catalysed reactions efficiently, with high activity and selectivity, during natural evolution. At the end of XIX century, the key-lock mechanism was proposed to explain the outstanding performance of enzymes. Specifically, close fitting of the substrate molecule (key) to the active centre of the enzyme (lock) was introduced to describe enzymatic properties. This simply and elegant theory works successfully in popular science until today, yet it fails when the active site is hidden deep within the protein core. Such enzymes, widely spread throughout the protein world, are equipped with tunnels possessing properties which can regulate the activity and selectivity of enzymes. These additional constraints provide an opportunity for control of reactivity and make enzymes with buried active sites ideal candidates for industrial and medical applications.

Most of the strategies proposed for enzyme redesign are focused on reengineering the vicinity of the active site. Such methods often result in loss of enzyme activity due to the rearrangement of residues that are crucial for the enzyme’s catalytic properties. Modification of the residues that build tunnels can provide a safe alternative to existing protein design protocols; however this requires a deep understanding of the transport phenomena through the tunnel network. Unfortunately due to the lack of experimental methods that can explore ligand transportation inside the protein core, the task of modifying tunnel residues is quite difficult to achieve.

To overcome challenges in this project we are employing a combination of modern computational tools which can visualize the protein exits and substrate entry points into the buried active site. State of the art methodology will be used to support experimentalists and to precisely describe ligand transportation phenomena. Due to the grouping of studied ligands into set of 30 different compounds we will identify subtle differences responsible for enzyme selectivity to aid our models.

Moreover to facilitate our research we have constructed an enzyme equipped with a switch located inside the tunnel. By changing reduction-oxidation conditions we are able to open or close the tunnel. This enzyme can be viewed as a prototype of a future enzyme in which the activity can be switched on/off on request or activated in a particular part of the living cell. During the project we will validate the possibility of our system grafting into other enzymes and the possibility of additional switch modifications to enhance control.

Our project is proceeding in cooperation with one of the best protein engineering groups – the Loschmidt Laboratory in Brno in Czech Republic – where most of the experimental work will be carried out.

Publication:

1. Raczyńska A. et al. Transient binding sites at the surface of haloalkane dehalogenase LinB as locations for fine-tuning enzymatic activity. PLoS ONE 2023 18(2), e0280776. doi: 10.1371/journal.pone.0280776 ARTICLE^{Open Access}
2. Mitusińska, K., Skalski, T. & Góra, A. Simple Selection Procedure to Distinguish between Static and Flexible Loops. Int J Mol Sci 21, 2293 (2020)
3. Mitusińska, K., Raczyńska, A., Bzówka, M., Bagrowska, W. & Góra, A. Applications of water molecules for analysis of macromolecule properties. Computational and Structural Biotechnology Journal 18, 355–365 (2020)
4. Magdziarz, T. et al. AQUA-DUCT 1.0: structural and functional analysis of macromolecules from an intramolecular voids perspective. Bioinformatics (2019). doi:10.1093/bioinformatics/btz946
5. Brezovsky, J. et al. Engineering a de Novo Transport Tunnel. ACS Catal 6, 7597–7610 (2016)

Project is financed by:

grant no. UMO-2015/18/M/NZ1/00427

Single Nucleotide Polymorphisms (SNPs) are DNA sequence variations that can lead to the changes in the amino acid sequence of the coded protein (nonsynonymous SNPs). Such mutations can result in changes that are well tolerated in organism. However, they might play important role in specific circumstances, e.g. during intensive clinical therapy, affecting patient response to the treatment.

The current research on genetic polymorphisms apply statistical approaches to existing databases of various experimental data predominantly. Various high-throughput sequencing methods were applied to identify non-synonymous variants in the human genome and several databases were constructed to collect and examine the relationship between human genome sequence variation and the associated disease phenotypes. As a consequence a huge number of bioinformatics approaches were developed to predict functional and structural consequences of the SNPs. The quality of such an approach and the accuracy of the results corresponds to the quality of the collected observables and the database uniformity.

In our project we are exploring parallel path of genetic polymorphisms study – we aim to investigate molecular basis of the clinically observed correlations in patient response to cancer treatment by deep analysis of the influence of particular mutations into functionality of proteins involved in drug metabolism. Our aim is to propose modifications in treatment scheme leading to personalised medicine based on individual genetic pattern of the patient.