Jiří Damborský

Masaryk University | MUNI · Department of Experimental Biology Loschmidt Laboratories

ChannelsDB 2.0 is an updated database providing structural information about the position, geometry and physicochemical properties of protein channels—tunnels and pores—within deposited biomacromolecular structures from PDB and AlphaFoldDB databases. The newly deposited information originated from several sources. Firstly, we included data calculat...

Haloalkane dehalogenases (HLDs) are a family of α/β-hydrolase fold enzymes that employ SN2 nucleophilic substitution to cleave the carbon–halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Atomic-level knowledge of both the inner organization and supramolecular complexation of HLDs is thus crucial...

Recent progress in engineering highly promising biocatalysts has increasingly involved machine learning methods. These methods leverage existing experimental and simulation data to aid in the discovery and annotation of promising enzymes, as well as in suggesting beneficial mutations for improving known targets. The field of machine learning for pr...

Enzymes enable sustainable and environmentally friendly solutions in industrial biocatalysis, bioremediation, and biosensing. Evolutionary data have proven pivotal for narrowing down the vast sequence space during enzyme optimization. However, capturing all important dependencies among residues is challenging due to the nonlinear influence of coevo...

ChannelsDB 2.0 is an updated database providing structural information about the position, geometry and physicochemical properties of protein channels - tunnels and pores - within deposited biomacromolecular structures from PDB and AlphaFoldDB databases. The newly deposited information originated from several sources. Firstly, we included data calc...

Polyethylene terephthalate (PET) is a widely used polyester due to its beneficial material properties and low cost. However, PET contributes significantly to the growing problem of plastic waste pollution. Enzymatic PET recycling has emerged as a promising alternative to conventional mechanical and chemical recycling methods. While many PET hydrola...

Thermostability is an essential requirement for the use of enzymes in the bioindustry. Here, we compare different protein stabilization strategies using a challenging target, a stable haloalkane dehalogenase DhaA115. We observe better performance of automated stabilization platforms FireProt and PROSS in designing multiple-point mutations over the...

Environmentally friendly industrial and biotech processes greatly benefit from enzyme‐based technologies. Their use is often possible only when the enzyme‐catalytic mechanism is thoroughly known. Thus, atomic‐level knowledge of a Michaelis enzyme‐substrate complex, revealing molecular details of substrate recognition and catalytic chemistry, is cru...

Recent efforts to develop comprehensive metabolome and proteome brain atlases for animal models have yielded significant progress. However, the ganglioside (GSs) profile of these models remains largely unexplored. As essential components of the brain, GSs play a crucial role in neuronal function. To address this gap in knowledge, we conducted an in...

Haloalkane dehalogenase (HLD) enzymes employ an SN2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well‐characterized enzymes, but a mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily...

Access pathways in enzymes are crucial for the passage of substrates and products of catalyzed reactions. The process can be studied by computational means with variable degrees of precision. Our in-house approximative method CaverDock provides a fast and easy way to set up and run ligand binding and unbinding calculations through protein tunnels a...

Background Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer’s Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. Methods Here, we reveal the molecular mechanism of ApoE4 aggregation using a co...

Nowadays, the vastly increasing demand for novel biotechnological products is supported by the continuous development of biocatalytic applications which provide sustainable green alternatives to chemical processes. The success of a biocatalytic application is critically dependent on how quickly we can identify and characterize enzyme variants fitti...

Cardiovascular diseases, such as myocardial infarction, ischemic stroke, and pulmonary embolism, are the most common causes of disability and death worldwide. Blood clot hydrolysis by thrombolytic enzymes and thrombectomy are key clinical interventions. The most widely used thrombolytic enzyme is alteplase, which has been used in clinical practice...

Intrinsic protein dynamics contribute to their biological functions. Rational engineering of protein dynamics is extremely challenging with only a handful of successful examples. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) represents a powerful technique for quantitative analysis of protein dynamics. Here we provide a detailed...

Tunnels in enzymes with buried active sites are key structural features allowing the entry of substrates and the release of products, thus contributing to the catalytic efficiency. Targeting the bottlenecks of protein tunnels is also a powerful protein engineering strategy. However, the identification of functional tunnels in multiple protein struc...

The choroid plexus (ChP) produces and is bathed in the cerebrospinal fluid (CSF), which in aging and Alzheimer's disease (AD) shows extensive proteomic alterations including evidence of inflammation. Considering inflammation hampers functions of the involved tissues, the CSF abnormalities reported in these conditions are suggestive of ChP injury. I...

Alzheimer's disease (AD) is characterized by the deposition of misfolded tau and amyloid-beta (Aβ). Tramiprosate (TMP) and its metabolite 3-sulfopropanoic acid (SPA) are phase 3 therapeutics believed to target Aβ oligomers. It is of paramount importance to understand how TMP/SPA modulate the conformations of Aβ. Here, we studied the Aβ42 alone and...

Protein engineering and synthetic biology are currently very active areas of research and development. In the pursuit of engineering proteins with specific capabilities, it has become evident that the scrutiny of structural and geometrical properties does not suffice to achieve the proposed goals. The dynamics and hydration of specific protein area...

The widely used coelenterazine-powered Renilla luciferase was discovered over 40 years ago, but the oxidative mechanism by which it generates blue photons remains unclear. Here we decipher Renilla-type catalysis through crystallographic, spectroscopic and computational experiments. Structures of ancestral and extant luciferases complexed with the s...

NanoLuc, a superior beta-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques were combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Binding to the al...

NanoLuc, a superior β-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques were combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Binding to the allos...

TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer...

Protein tunnels play an essential role in transporting small molecules into the active sites of enzymes. Tunnels' geometrical and physico-chemical properties influence the transport process. The tunnels are attractive hot spots for protein engineering and drug development. However, studying the ligand binding and unbinding using experimental techni...

Background Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer’s Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. Methods Here, we reveal the molecular mechanism of ApoE4 aggregation using a co...

Many dynamic interactions within the cell microenvironment modulate cell behavior and cell fate. However, the pathways and mechanisms behind cell-cell or cell-extracellular matrix interactions remain understudied, as they occur at a nanoscale level. Recent progress in nanotechnology allows for mimicking of the microenvironment at nanoscale in vitro...

Next-generation sequencing doubles genomic databases every 2.5 years. The accumulation of sequence data provides a unique opportunity to identify interesting biocatalysts directly in the databases without tedious and time-consuming engineering. Herein, we present a pipeline integrating sequence and structural bioinformatics with microfluidic enzymo...

Stroke burden is substantially increasing but current therapeutic drugs are still far from ideal. Here we highlight the vast potential of staphylokinase as an efficient, fibrin-selective, inexpensive, and evolvable thrombolytic agent. The emphasis is escalated by new recent findings. Staphylokinase nonimmunogenic variant was proven noninferior to a...

Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metag...

Although the link between microbial infections and Alzheimer's disease (AD) has been demonstrated in multiple studies, the involvement of pathogens in the development of AD remains unclear. Here, we investigated the frequency of the 10 most commonly cited viral and bacterial pathogens in serum and cerebrospinal fluid (CSF) of AD patients. We have u...

Although flavin-dependent halogenases (FDHs) are attractive for C–H bond activation, their applications are limited due to low turnover and stability. We have previously shown that leakage of a halogenating intermediate, hypohalous acid (HOX), causes FDHs to be inefficient by lessening halogenation yield. Here we employed a mechanism-guided semi-ra...

Acceleration of chemical reactions by the enzymes optimized using protein engineering represents one of the key pillars of biotechnology's contribution toward sustainability. Tunnels and channels of enzymes with buried active sites enable the exchange of ligands, ions, and water molecules between the outer environment and active site pockets. The e...

Fluorinases, the only enzymes known to catalyze the transfer of fluorine to an organic molecule, are essential catalysts for the biological synthesis of valuable organofluorines. However, the few fluorinases identified so far have low turnover rates that hamper biotechnological applications. Here, we isolated and characterized putative fluorinases...

HaloTag labeling technology has introduced unrivaled potential in protein chemistry and molecular and cellular biology. A wide variety of ligands have been developed to meet the specific needs of diverse applications, but only a single protein tag, DhaAHT, is routinely used for their incorporation. Following a systematic kinetic and computational a...

The importance of the quantitative description of protein unfolding and aggregation for the rational design of stability or understanding the molecular basis of protein misfolding diseases is well established. Protein thermostability is typically assessed by calorimetric or spectroscopic techniques that monitor different complementary signals durin...

Next-generation sequencing doubles genomic databases every 2.5 years. The accumulation of sequence data provides a unique opportunity to identify interesting biocatalysts directly in the databases without tedious and time-consuming engineering. Herein, we present a pipeline integrating sequence and structural bioinformatics with microfluidic enzymo...

Next-generation sequencing doubles genomic databases every 2.5 years. The accumulation of sequence data provides a unique opportunity to identify interesting biocatalysts directly in the databases without tedious and time-consuming engineering. Herein, we present a pipeline integrating sequence and structural bioinformatics with microfluidic enzymo...

The transplantation of loops between structurally related proteins is a compelling method to improve the activity, specificity and stability of enzymes. However, despite the interest of loop regions in protein engineering, the available methods of loop-based rational protein design are scarce. One particular difficulty related to loop engineering i...

Fluorinases, the only enzymes known to catalyze the transfer of fluorine to an organic molecule, are essential biocatalysts for the sustainable synthesis of valuable organofluorines. However, the few fluorinases identified so far have low turnover rates that hamper biotechnological applications. Here, we isolated and characterized putative fluorina...

The Front Cover shows an alkylamine molecule about to enter an enzyme through its substrate tunnel, which is currently under construction. In their Research Article, L. R. Rapp et al. describe dodecylamine as a P450 wild‐type inhibitor that could be accepted as a novel substrate by engineering the enzyme's entrance tunnel using a simple MS‐based ra...

Cardio- and cerebrovascular diseases are leading causes of death and disability, resulting in one of the highest socio-economic burdens of any disease type. The discovery of bacterial and human plasminogen activators and their use as thrombolytic drugs have revolutionized treatment of these pathologies. Fibrin-specific agents have an advantage over...

Polyethylene terephthalate (PET) is the most widespread synthetic polyester, having been utilized in textile fibers and packaging materials for beverages and food, contributing considerably to the global solid waste stream and environmental plastic pollution. While enzymatic PET recycling and upcycling have recently emerged as viable disposal metho...

The widely used coelenterazine-powered Renilla luciferase was discovered over 40 years ago but the oxidative mechanism by which it generates blue photons remains unclear. Here we decipher Renilla-type bioluminescence through crystallographic, spectroscopic, and computational experiments. Structures of ancestral and extant luciferases complexed with...

Therapeutic enzymes are valuable biopharmaceuticals in various biomedical applications. They have been successfully applied for fibrinolysis, cancer treatment, enzyme replacement therapies, and the treatment of rare diseases. Still, there is a permanent demand to find new or better therapeutic enzymes, which would be sufficiently soluble, stable, a...

The catalytic space of the P450 monooxygenase CYP153AM.aq was opened from a terminal (ω‐) fatty acid hydroxylase to a catalyst capable of performing ω‐hydroxylation of dodecylamine, which is a potent inhibitor for the wild‐type enzyme. A simple screening method named Rapid‐flow Analysis of Product Peaks (RAPP) was established and applied to measure...

Aging and Alzheimer’s disease (AD), a major age-related disorder, are both characterized by inflammatory changes in the cerebrospinal fluid (CSF). The origin and the mechanisms underlying these inflammatory changes, however, remain poorly understood. Here, we report that aging elicits inflammatory changes in the CSF that become accentuated uniquely...

HaloTag labeling technology has introduced unrivaled potential in protein chemistry, molecular and cellular biology. A wide variety of ligands have been developed to meet the specific needs of diverse applications, but only a single protein tag, DhaAHT, is routinely used for their incorporation. Following a systematic kinetic and computational anal...

The Protein Data Bank in Europe – Knowledge Base (PDBe-KB, https://pdbe-kb.org) is an open collaboration between world-leading specialist data resources contributing functional and biophysical annotations derived from or relevant to the Protein Data Bank (PDB). The goal of PDBe-KB is to place macromolecular structure data in their biological contex...

Objective Although the link between microbial infections and Alzheimer’s disease (AD) has been demonstrated in multiple studies, the involvement of pathogens in the development of AD remains unclear. Therefore, this theory beckons further systematic investigation. In this study, we have examined the association between the 10 most widely discussed...

Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstructio...

Next-generation sequencing doubles genomic databases every 2.5 years. The accumulation of sequence data raises the need to speed up functional analysis. Herein, we present a pipeline integrating bioinformatics and microfluidics and its application for high-throughput mining of novel haloalkane dehalogenases. We employed bioinformatics to identify 2...

The plasminogen activator staphylokinase is a fibrin-specific thrombolytic biomolecule and an attractive target for the development of effective myocardial infarction and stroke therapy. To engineer the protein rationally, a detailed understanding of the biochemical mechanism and limiting steps is essential. Conventional fitting to equations derive...

The plasminogen activator staphylokinase is a fibrin-specific thrombolytic biomolecule and an attractive target for the development of effective myocardial infarction and stroke therapy. To engineer the protein rationally, a detailed understanding of the biochemical mechanism and limiting steps is essential. Conventional fitting to equations derive...

Ionic liquids attracted interest as green alternatives to replace conventional organic solvents in protein stability studies. They can play an important role in the stabilization of enzymes such as haloalkane dehalogenases that are used for biodegradation of warfare agents and halogenated environmental pollutants. Three-dimensional crystals of halo...

The functionality of a protein depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology appl...

Enzymes are in high demand for very diverse biotechnological applications. However, natural biocatalysts often need to be engineered for fine-tuning their properties towards the end applications, such as the activity, selectivity, stability to temperature or co-solvents, and solubility. Computational methods are increasingly used in this task, prov...

Tacrine is a classic drug whose efficacy against neurodegenerative diseases is still shrouded in mystery. It seems that besides its inhibitory effect on cholinesterases, the clinical benefit is co-determined by NMDAR-antagonizing activity. Our previous data showed that the direct inhibitory effect of tacrine, as well as its 7-methoxy derivative (7-...

The development of microbial products for cancer treatment has been in the spotlight in recent years. In order to accelerate the lengthy and expensive drug development process, in silico screening tools are systematically employed, especially during the initial discovery phase. Moreover, considering the steadily increasing number of molecules appro...

Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein Anc HLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of Anc H...

Haloalkane dehalogenases and epoxide hydrolases are phylogenetically related and structurally homologous enzymes that use nucleophilic aspartate residues for an SN2 attack on their substrates. Despite their mechanistic similarities, no enzymes are known that exhibit both epoxide hydrolase and dehalogenase activity. We screened a subset of epoxide h...

The new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes pathological pulmonary symptoms. Most efforts to develop vaccines and drugs against this virus target the spike glycoprotein, particularly its S1 subunit, which is recognised by angiotensin-converting enzyme 2. Here we use the in-house developed tool CaverDock to perform vi...

Haloalkane dehalogenases (EC 3.8.1.5) are microbial enzymes that catalyse the hydrolytic conversion of halogenated compounds, resulting in a halide ion, a proton and an alcohol. These enzymes are used in industrial biocatalysis, bioremediation and biosensing of environmental pollutants or for molecular tagging in cell biology. The novel haloalkane...

The ability to engineer enzymes for industrial and biomedical applications is primarily limited by a paucity of mechanistic understanding. To gain insight into the mechanisms of enzyme catalysis, one must screen enormous numbers of discrete reaction conditions, which is a laborious task using conventional technologies. To address such limitations,...

Cytochrome P450 CYP153A M.aq from Marinobacter aquaeolei serves as a model enzyme for the terminal (ω-) hydroxylation of medium-to long-chain fatty acids. We have engineered this enzyme using different mutagenesis approaches based on structure-sequence-alignments within the 3DM database and crystal structures of CYP153A M.aq and a homologue CYP153A...

The development of microbial products for cancer treatment has been in the spotlight in recent years. In order to accelerate the lengthy and expensive drug development process, in silico screening tools are systematically employed, especially during the initial discovery phase. Moreover, considering the steadily increasing number of molecules appro...

Protein evolution and protein engineering techniques are of great interest in basic science and industrial applications such as pharmacology, medicine, or biotechnology. Ancestral sequence reconstruction (ASR) is a powerful technique for probing evolutionary relationships and engineering robust proteins with good thermostability and broad substrate...