Jan Brezovsky

Adam Mickiewicz University in Poznań | UAM · Institute of Molecular Biology and Biotechnology

Enzymes are pivotal to numerous biological processes, often featuring buried active sites linked to the surrounding solvent through intricate and dynamic tunnels. These tunnels are vital for facilitating substrate access, enabling product release, and regulating solvent exchange, which collectively influence enzymatic function and efficiency. Conse...

An aqueous environment is vital for life as we know it, and water is essential for nearly all biochemical processes at the molecular level. Proteins utilize water molecules in various ways. Consequently, proteins must transport water molecules across their internal network of tunnels to reach the desired action sites, either within them or by funct...

ABCG46 of the legume Medicago truncatula is an ABC-type transporter responsible for highly selective translocation of the phenylpropanoids, 4-coumarate, and liquiritigenin, over the plasma membrane. To investigate molecular determinants of the observed substrate selectivity, we applied a combination of phylogenetic and biochemical analyses, AlphaFo...

Information regarding pathways through voids in biomolecules and their roles in ligand transport is critical to our understanding of the function of many biomolecules. Recently, the advent of high-throughput molecular dynamics simulations has enabled the study of these pathways, and of rare transport events. However, the scale and intricacy of the...

Protein hydration plays a vital role in many biological functions, and molecular dynamics simulations are frequently used to study it. However, the accuracy of these simulations is often sensitive to the water model used, a phenomenon particularly evident in intrinsically disordered proteins. Here, we investigated the extent to which the choice of...

Tunnels are structural conduits in biomolecules responsible for transporting chemical compounds and solvent molecules from the active site. They have been shown to be present in a wide variety of enzymes across all functional and structural classes. However, the study of such pathways is experimentally challenging, because they are typically transi...

Because most proteins have buried active sites, protein tunnels or channels play a crucial role in the transport of small molecules into buried cavities for enzymatic catalysis. Tunnels can critically modulate the biological process of protein–ligand recognition. Various molecular dynamics methods have been developed for exploring and exploiting th...

Tunnels are structural conduits in biomolecules responsible for transporting chemical compounds and solvent molecules to and from the active site. Tunnels have been shown to be present in a wide variety of enzymes across all their functional and structural classes. However, the study of such pathways is experimentally challenging, as they are typic...

Experimental approaches for identifying protein structures and conducting their analyses face systemic limitations and challenges. Thus, computational approaches have emerged as invaluable tools over the past few decades, offering complementary insights into the protein structure, function and analysis. This chapter provides a focused overview of c...

Given that most proteins have buried active sites, protein tunnels or channels play a crucial role in mitigating the transport of small molecules to the buried cavity for enzymatic catalysis. Tunnels can critically modulate the biological process of protein-ligand recognition. Various molecular dynamics methods have been developed for exploring and...

Understanding the utilization of tunnels and water transport within enzymes is crucial for the catalytic function of enzymes, as water molecules can stabilize bound substrates and help with unbinding processes of products and inhibitors. Since the choice of water models for molecular dynamics simulations was shown to determine the accuracy of vario...

An aqueous environment is necessary for life as we know it, and water is required for almost all biochemical processes at a molecular level. Proteins employ water molecules in many ways. Hence, proteins need to transport water molecules across their internal network of tunnels to reach the desired action sites, either inside them or acting as molec...

The rapid rise of antibiotic-resistant bacteria necessitates the search for alternative, unconventional solutions, such as targeting bacterial communication. Signal disruption can be achieved by enzymatic degradation of signaling compounds, reducing the expression of genes responsible for virulence, biofilm formation, and drug resistance while evad...

Protein hydration plays a vital role in many biological functions and molecular simulations are frequently used to study the effect of protein hydration at the atomic level. However, the accuracy of these simulations has often been highly sensitive to the water model used, a phenomenon best known in the case of intrinsically disordered proteins. In...

Green and nano-structured catalytic media are vital for bio-catalysis to attenuate the denaturation tendency of biocatalysts under severe reaction conditions. Hydrotropes with multi-faceted physiochemical properties represent promising systems for sustainable protein packaging. Herein, the suitability of adenosine-5’-triphosphate (ATP) and choliniu...

Green and nano-structured catalytic media are vital for bio-catalysis to attenuate the denaturation tendency of biocatalysts under severe reaction conditions. Hydrotropes with multi-faceted physiochemical properties represent promising systems for sustainable...

Nowadays, molecular dynamics (MD) simulations of proteins with hundreds of thousands of snapshots are commonly produced using modern GPUs. However, due to the abundance of data, analyzing transport tunnels present in the internal voids of these molecules, in all generated snapshots, has become challenging. Here, we propose to combine the usage of C...

Brief outline of the methodology to systematically detect transport events through tunnels and benchmark with different water models. Poster also explains how water can squeeze into narrow tunnels to reach the active site and vice versa. Also some results included from TransportTools.

ABCG46 of the legume Medicago truncatula is an ABC-type plasma membrane transporter that selectively translocates endogenous phenylpropanoids from the biosynthetic pathway of the phytoalexin, medicarpin, namely 4-coumarate and liquiritigenin. To investigate molecular determinants of the observed substrate selectivity, we applied a combination of ph...

ABCG46 of the legume Medicago truncatula is an ABC-type plasma membrane transporter that selectively translocates endogenous phenylpropanoids from the biosynthetic pathway of the phytoalexin, medicarpin, namely 4-coumarate and liquiritigenin. To investigate molecular determinants of the observed substrate selectivity, we applied a combination of ph...

Haloalkane dehalogenase is an environmentally important biocatalyst that catalyzes the hydrolysis of the carbon-halogen bond. These are hydrolytic enzymes, requiring the presence of water molecules in their active site to perform their action. The active site of these enzymes is deeply buried, meaning that the substrate and water molecules have to...

Due to the alarming global crisis of the growing microbial antibiotic resistance, investigation of alternative strategies to combat this issue has gained considerable momentum in the recent decade. A quorum quenching (QQ) process disrupts bacterial communication through so-called quorum sensing that enables bacteria to sense the cell density in the...

Hydrolytic enzymes require the presence of water molecules in their active sites to perform their action. However, many enzymes have their active sites buried, which requires molecules of their cognate ligands and water to travel through transport pathways, called tunnels, to reach these sites. Since the tunnels relevant for water transport are oft...

Information regarding pathways through voids in biomolecules and their roles in ligand transport is critical to our understanding of the function of many biomolecules. Recently, the advent of high-throughput molecular dynamics simulations has enabled the study of these pathways, and of rare transport events. However, the scale and intricacy of the...

Progress in technology and algorithms throughout the past decade has transformed the field of protein design and engineering. Computational approaches have become well-engrained in the processes of tailoring proteins for various biotechnological applications. Many tools and methods are developed and upgraded each year to satisfy the increasing dema...

Computational prediction has become an indispensable aid in the processes of engineering and designing proteins for various biotechnological applications. With the tremendous progress in more powerful computer hardware and more efficient algorithms, some of in silico tools and methods have started to apply the more realistic description of proteins...

Haloalkane dehalogenases are enzymes with a broad application potential in biocatalysis, bioremediation, biosensing and cell imaging. The new haloalkane dehalogenase DmxA originating from the psychrophilic bacterium Marinobacter sp. ELB17 surprisingly possesses the highest thermal stability (apparent melting temperature Tm,app = 65.9 °C) of all bio...

Caver Web 1.0 is a web server for comprehensive analysis of protein tunnels and channels, and study of the ligands' transport through these transport pathways. Caver Web is the first interactive tool allowing both the analyses within a single graphical user interface. The server is built on top of the abundantly used tunnel detection tool Caver 3.0...

Motivation: Protein tunnels and channels are key transport pathways that allow ligands to pass between proteins' external and internal environments. These functionally important structural features warrant detailed attention. It is difficult to study the ligand binding and unbinding process experimentally, while molecular dynamics simulations can...

Here we present a novel method for the analysis of transport processes in proteins and its implementation called CaverDock. Our method is based on a modified molecular docking algorithm. It iteratively places the ligand along the access tunnel in such a way that the ligand movement is contiguous and the energy is minimized. The result of CaverDock...

Enzyme engineering tends to focus on the design of active sites for the chemical steps, while the physical steps of the catalytic cycle are often overlooked. Tight binding of a substrate in an active site is beneficial for the chemical steps, whereas good accessibility benefits substrate binding and product release. Many enzymes control the accessi...

Here we present a novel method for the analysis of transport processes in proteins and its implementation called CaverDock. Our method is based on a modified molecular docking algorithm. It iteratively places the ligand along the access tunnel in such a way that the ligand movement is contiguous and the energy is minimized. The result of CaverDock...

Stability is one of the most important characteristics of proteins employed as biocatalysts, biotherapeutics and biomaterials, and the role of computational approaches in modifying protein stability is rapidly expanding. We have recently identified stabilizing mutations in haloalkane dehalogenase DhaA using phylogenetic analysis but were not able t...

Understanding the protein-ligand interactions is crucial for drug design, enzymology research or enzyme engineering. The interaction of a protein and a ligand molecule often takes place in protein's binding or active site. Such functional sites may be hidden inside the protein core, and therefore a transport of a ligand from outside environment to...

Motivation Studying the transport paths of ligands, solvents, or ions in transmembrane proteins and proteins with buried binding sites is fundamental to the understanding of their biological function. A detailed analysis of the structural features influencing the transport paths is also important for engineering proteins for biomedical and biotechn...

Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Here, we describe an integrated system for automated in silico screening and systematic characterization of diverse family members. The workflow consists of (i) identification and computational characterization of relevant...

The traditional way of rationally engineering enzymes to change their biocatalytic properties utilizes the modifications of their active sites. Another emerging approach is the engineering of structural features involved in the exchange of ligands between buried active sites and the surrounding solvent. However, surprisingly little is known about t...

Protein tunnels connecting the functional buried cavities with bulk solvent and protein channels, enabling the transport through biological membranes, represent the structural features that govern the exchange rates of ligands, ions, and water solvent. Tunnels and channels are present in a vast number of known proteins and provide control over thei...

Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer-assi...

The anthropogenic toxic compound 1,2,3-trichloropropane is poorly degradable by natural enzymes. We have previously constructed the haloalkane dehalogenase DhaA31 by focused directed evolution (Pavlova, M. et al., Nat. Chem. Biol. 2009, 5, 727–733), which is 32 times more active than the wild-type enzyme and is currently the most active variant kno...

The inside back cover picture shows the reconstruction of the ancestral sequence of haloalkane dehalogenases. Ancestral enzymes exhibited significantly enhanced thermodynamic stability compared to extant ones, as well as higher specific activities with a preference for short, multisubstituted halogenated substrates. Ancestral sequence reconstructio...

There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stabilit...

Ancestral sequence reconstruction (ASR) represents a powerful approach for empirical testing structure-function relationships of diverse proteins. We employed ASR to predict sequences of five ancestral haloalkane dehalogenases (HLDs) from the HLD-II subfamily. Genes encoding the inferred ancestral sequences were synthesized and expressed in Escheri...

The enzymatic enantiodiscrimination of linear β-haloalkanes is difficult because the simple structures of the substrates prevent directional interactions. Herein we describe two distinct molecular mechanisms for the enantiodiscrimination of the β-haloalkane 2-bromopentane by haloalkane dehalogenases. Highly enantioselective DbjA has an open, solven...

Fibroblast growth factors (FGFs) play numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability in vivo and in vitro . Here we tackle this problem using a genera...

Many enzymes contain tunnels and gates that are essential to their function. Gates reversibly switch between open and closed conformations and thereby control the traffic of small molecules-substrates, products, ions, and solvent molecules-into and out of the enzyme's structure via molecular tunnels. Many transient tunnels and gates undoubtedly rem...

Penicillin G acylase is one of the most employed enzymes in the pharmaceutical industry due to its role in the biotransformation of semi-synthetic β-lactam antibiotics. Recently, the enantioselectivity of the penicillin G acylase markedly broadened its application potential. In this study, we have evaluated effects of in-silico replacements of acyl...

Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. Absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional ?hole? into the protein represent...

An important message taken from human genome sequencing projects is that the human population exhibits approximately 99.9% genetic similarity. Variations in the remaining parts of the genome determine our identity, trace our history and reveal our heritage. The precise delineation of phenotypically causal variants plays a key role in providing accu...

HotSpot Wizard 2.0 is a web server for automated identification of hot spots and design of smart libraries for engineering proteins’ stability, catalytic activity, substrate specificity and enantioselectivity. The server integrates sequence, structural and evolutionary information obtained from 3 databases and 20 computational tools. Users are guid...

Haloalkane dehalogenases (HLDs) have recently been discovered in a number of bacteria, including symbionts and pathogens of both plants and humans. However, the biological roles of HLDs in these organisms are unclear. The development of efficient HLD inhibitors serving as molecular probes to explore their function would represent an important step...

Life sciences are yielding huge data sets that underpin scientific discoveries fundamental to improvement in human health, agriculture and the environment. In support of these discoveries, a plethora of databases and tools are deployed, in technically complex and diverse implementations, across a spectrum of scientific disciplines. The corpus of do...

There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagoni...

Penicillin G acylases (PGAs) are enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates. Among them, derivatives of α- and β-amino acids represent a class of compounds with high application potential. PGAEc from Escherichia coli and PGAA from Achromobacter sp. CCM 4824 were used to catalyze enantiose...

Wedelolactone, a plant coumestan, was shown to act as anti-cancer agent for breast and prostate carcinomas in vitro and in vivo targeting multiple cellular proteins including androgen receptors, 5-lipoxygenase and topoisomerase IIα. It is cytotoxic to breast, prostate, pituitary and myeloma cancer cell lines in vitro at μM concentrations. In this s...

Hydration of proteins profoundly affects their functions. We describe a simple and general method for site-specific analysis of protein hydration based on the in vivo incorporation of fluorescent unnatural amino acids and their analysis by steady-state fluorescence spectroscopy. Using this method, we investigate the hydration of functionally import...

A variant of the haloalkane dehalogenase DhaA with greatly enhanced stability and tolerance of organic solvents but reduced activity was created by mutating four residues in the access tunnel. To create a stabilised enzyme with superior catalytic activity, two of the four originally modified residues were randomised. The resulting mutant F 176 G ex...

The biological function of a macromolecule often requires that a small molecule or ion is transported through its structure. The transport pathway often leads through void spaces in the structure. The properties of transport pathways change significantly in time; therefore, the analysis of a trajectory from molecular dynamics rather than of a singl...

The substrate specificity is a key feature of enzymes determining their applicability in biomaterials and biotechnologies. Experimental testing of activities with novel substrates is a time-consuming and inefficient process, typically resulting in many failures. Here, we present an experimentally validated in silico method for the discovery of nove...

Multienzyme processes represent an important area of biocatalysis. Their efficiency can be enhanced by optimization of the stoichiometry of the biocatalysts. Here we present a workflow for maximizing the efficiency of a three-enzyme system catalyzing a five-step chemical conversion. Kinetic models of pathways with wild-type or engineered enzymes we...

Traditional directed evolution experiments are often time-, labor- and cost-intensive because they involve repeated rounds of random mutagenesis and the selection or screening of large mutant libraries. The efficiency of directed evolution experiments can be significantly improved by targeting mutagenesis to a limited number of hot-spot positions a...

The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is uniq...

The transport of ligands, ions or solvent molecules into proteins with buried binding sites or through the membrane is enabled by protein tunnels and channels. CAVER Analyst is a software tool for calculation, analysis and real-time visualization of access tunnels and channels in static and dynamic protein structures. It provides an intuitive graph...