Haloalkane dehalogenase (HLD) enzymes employ an SN 2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD-like enzyme from the archaeon Haloferax mediterranei, by combining cryo-electron microscopy (cryo-EM) and x-ray crystallography. We show that full-length wild-type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring-like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three-dimensional reconstructions of an oligomeric species by single-particle cryo-EM. Moreover, we engineered a crystallizable mutant (DhmeAΔGG ) that provided diffraction-quality crystals. The 3.3 Å crystal structure reveals that DhmeAΔGG forms a ring-like 20-mer structure with outer and inner diameter of ~200 and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization, and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates.

• Keywords
• DhmeA, Haloferax mediterranei, catalysis, cryo-EM, haloalkane dehalogenase, multimerization, x-ray crystallography,
• MeSH
• Cryoelectron Microscopy methods   MeSH
• Hydrolases * chemistry   MeSH
• Crystallography, X-Ray MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• haloalkane dehalogenase MeSH Browser
• Hydrolases * MeSH

Computational design of protein catalysts with enhanced stabilities for use in research and enzyme technologies is a challenging task. Using force-field calculations and phylogenetic analysis, we previously designed the haloalkane dehalogenase DhaA115 which contains 11 mutations that confer upon it outstanding thermostability (T m = 73.5 °C; ΔT m > 23 °C). An understanding of the structural basis of this hyperstabilization is required in order to develop computer algorithms and predictive tools. Here, we report X-ray structures of DhaA115 at 1.55 Å and 1.6 Å resolutions and their molecular dynamics trajectories, which unravel the intricate network of interactions that reinforce the αβα-sandwich architecture. Unexpectedly, mutations toward bulky aromatic amino acids at the protein surface triggered long-distance (∼27 Å) backbone changes due to cooperative effects. These cooperative interactions produced an unprecedented double-lock system that: (i) induced backbone changes, (ii) closed the molecular gates to the active site, (iii) reduced the volumes of the main and slot access tunnels, and (iv) occluded the active site. Despite these spatial restrictions, experimental tracing of the access tunnels using krypton derivative crystals demonstrates that transport of ligands is still effective. Our findings highlight key thermostabilization effects and provide a structural basis for designing new thermostable protein catalysts.

• Publication type
• Journal Article MeSH

Haloalkane dehalogenases are enzymes that catalyze the cleavage of carbon-halogen bonds in halogenated compounds. They serve as model enzymes for studying structure-function relationships of >100.000 members of the α/β-hydrolase superfamily. Detailed kinetic analysis of their reaction is crucial for understanding the reaction mechanism and developing novel concepts in protein engineering. Fluorescent substrates, which change their fluorescence properties during a catalytic cycle, may serve as attractive molecular probes for studying the mechanism of enzyme catalysis. In this work, we present the development of the first fluorescent substrates for this enzyme family based on coumarin and BODIPY chromophores. Steady-state and pre-steady-state kinetics with two of the most active haloalkane dehalogenases, DmmA and LinB, revealed that both fluorescent substrates provided specificity constant two orders of magnitude higher (0.14-12.6 μM-1 s-1) than previously reported representative substrates for the haloalkane dehalogenase family (0.00005-0.014 μM-1 s-1). Stopped-flow fluorescence/FRET analysis enabled for the first time monitoring of all individual reaction steps within a single experiment: (i) substrate binding, (ii-iii) two subsequent chemical steps and (iv) product release. The newly introduced fluorescent molecules are potent probes for fast steady-state kinetic profiling. In combination with rapid mixing techniques, they provide highly valuable information about individual kinetic steps and mechanism of haloalkane dehalogenases. Additionally, these molecules offer high specificity and efficiency for protein labeling and can serve as probes for studying protein hydration and dynamics as well as potential markers for cell imaging.

• Keywords
• Enzyme kinetics, Fluorescent substrate, Haloalkane dehalogenase, Mechanism, Protein labeling,
• Publication type
• Journal Article MeSH

Transport of ligands between bulk solvent and the buried active sites is a critical event in the catalytic cycle of many enzymes. The rational design of transport pathways is far from trivial due to the lack of knowledge about the effect of mutations on ligand transport. The main and an auxiliary tunnel of haloalkane dehalogenase LinB have been previously engineered for improved dehalogenation of 1,2-dibromoethane (DBE). The first chemical step of DBE conversion was enhanced by L177W mutation in the main tunnel, but the rate-limiting product release was slowed down because the mutation blocked the main access tunnel and hindered protein dynamics. Three additional mutations W140A + F143L + I211L opened-up the auxiliary tunnel and enhanced the product release, making this four-point variant the most efficient catalyst with DBE. Here we study the impact of these mutations on the catalysis of bulky aromatic substrates, 4-(bromomethyl)-6,7-dimethoxycoumarin (COU) and 8-chloromethyl-4,4'-difluoro-3,5-dimethyl-4-bora-3a,4a-diaza-s-indacene (BDP). The rate-limiting step of DBE conversion is the product release, whereas the catalysis of COU and BDP is limited by the chemical step. The catalysis of COU is mainly impaired by the mutation L177W, whereas the conversion of BDP is affected primarily by the mutations W140A + F143L + I211L. The combined computational and kinetic analyses explain the differences in activities between the enzyme-substrate pairs. The effect of tunnel mutations on catalysis depends on the rate-limiting step, the complementarity of the tunnels with the substrates and is clearly specific for each enzyme-substrate pair.

• Keywords
• BDP, 8-chloromethyl-3,5-dimethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, COU, 4-(bromomethyl)-6,7-dimethoxycoumarin, Enzyme kinetics, Enzyme mutation, MD, molecular dynamics, MSM, Markov state model, NAC, near-attack conformer, Substrate specificity,
• Publication type
• Journal Article MeSH

Optofluidics, a research discipline combining optics with microfluidics, currently aspires to revolutionize the analysis of biological and chemical samples, e.g., for medicine, pharmacology, or molecular biology. In order to detect low concentrations of analytes in water, we have developed an optofluidic device containing a nanostructured substrate for surface enhanced Raman spectroscopy (SERS). The geometry of the gold surface allows localized plasmon oscillations to give rise to the SERS effect, in which the Raman spectral lines are intensified by the interaction of the plasmonic field with the electrons in the molecular bonds. The SERS substrate was enclosed in a microfluidic system, which allowed transport and precise mixing of the analyzed fluids, while preventing contamination or abrasion of the highly sensitive substrate. To illustrate its practical use, we employed the device for quantitative detection of persistent environmental pollutant 1,2,3-trichloropropane in water in submillimolar concentrations. The developed sensor allows fast and simple quantification of halogenated compounds and it will contribute towards the environmental monitoring and enzymology experiments with engineered haloalkane dehalogenase enzymes.

• Keywords
• 1,2,3-trichloropropane, Klarite 312, chloroalkane, microfluidics, surface enhanced Raman spectroscopy,
• Publication type
• Journal Article MeSH

The haloalkane dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized haloalkane dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications.IMPORTANCE We present a thorough biochemical characterization of the haloalkane dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols.

• Keywords
• biotechnology, cosolvents, enzyme, haloalkane dehalogenase, marine, microbial, stability, substrate specificity,
• MeSH
• Bacteria enzymology   genetics   metabolism   MeSH
• Biocatalysis MeSH
• Biotechnology MeSH
• Hydrolases chemistry   genetics   isolation & purification   metabolism   MeSH
• Catalytic Domain MeSH
• Catalysis MeSH
• Kinetics MeSH
• Hydrogen-Ion Concentration MeSH
• Crystallization MeSH
• Metagenome MeSH
• Microbial Consortia genetics   physiology   MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH

The computational prediction of unbinding rate constants is presently an emerging topic in drug design. However, the importance of predicting kinetic rates is not restricted to pharmaceutical applications. Many biotechnologically relevant enzymes have their efficiency limited by the binding of the substrates or the release of products. While aiming at improving the ability of our model enzyme haloalkane dehalogenase DhaA to degrade the persistent anthropogenic pollutant 1,2,3-trichloropropane (TCP), the DhaA31 mutant was discovered. This variant had a 32-fold improvement of the catalytic rate toward TCP, but the catalysis became rate-limited by the release of the 2,3-dichloropropan-1-ol (DCP) product from its buried active site. Here we present a computational study to estimate the unbinding rates of the products from DhaA and DhaA31. The metadynamics and adaptive sampling methods were used to predict the relative order of kinetic rates in the different systems, while the absolute values depended significantly on the conditions used (method, force field, and water model). Free energy calculations provided the energetic landscape of the unbinding process. A detailed analysis of the structural and energetic bottlenecks allowed the identification of the residues playing a key role during the release of DCP from DhaA31 via the main access tunnel. Some of these hot-spots could also be identified by the fast CaverDock tool for predicting the transport of ligands through tunnels. Targeting those hot-spots by mutagenesis should improve the unbinding rates of the DCP product and the overall catalytic efficiency with TCP.

• Keywords
• CaverDock, adaptive sampling, metadynamics, molecular dynamics, protein engineering, unbinding kinetics,
• Publication type
• Journal Article MeSH

ChannelsDB (http://ncbr.muni.cz/ChannelsDB) is a database providing information about the positions, geometry and physicochemical properties of channels (pores and tunnels) found within biomacromolecular structures deposited in the Protein Data Bank. Channels were deposited from two sources; from literature using manual deposition and from a software tool automatically detecting tunnels leading to the enzymatic active sites and selected cofactors, and transmembrane pores. The database stores information about geometrical features (e.g. length and radius profile along a channel) and physicochemical properties involving polarity, hydrophobicity, hydropathy, charge and mutability. The stored data are interlinked with available UniProt annotation data mapping known mutation effects to channel-lining residues. All structures with channels are displayed in a clear interactive manner, further facilitating data manipulation and interpretation. As such, ChannelsDB provides an invaluable resource for research related to deciphering the biological function of biomacromolecular channels.

• MeSH
• Amino Acids chemistry   metabolism   MeSH
• Cytochrome P-450 CYP2D6 chemistry   genetics   metabolism   MeSH
• Databases, Protein * MeSH
• Eukaryotic Cells cytology   enzymology   MeSH
• Gene Expression MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Ion Channels chemistry   genetics   metabolism   MeSH
• Nuclear Pore chemistry   genetics   metabolism   MeSH
• Catalytic Domain MeSH
• Coenzymes chemistry   metabolism   MeSH
• Humans MeSH
• Mutation MeSH
• Prokaryotic Cells cytology   enzymology   MeSH
• Software * MeSH
• Static Electricity MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids MeSH
• Cytochrome P-450 CYP2D6 MeSH
• Ion Channels MeSH
• Coenzymes MeSH

Studies of protein unfolding mechanisms are critical for understanding protein functions inside cells, de novo protein design as well as defining the role of protein misfolding in neurodegenerative disorders. Calorimetry has proven indispensable in this regard for recording full energetic profiles of protein unfolding and permitting data fitting based on unfolding pathway models. While both kinetic and thermodynamic protein stability are analysed by varying scan rates and reheating, the latter is rarely used in curve-fitting, leading to a significant loss of information from experiments. To extract this information, we propose fitting both first and second scans simultaneously. Four most common single-peak transition models are considered: (i) fully reversible, (ii) fully irreversible, (iii) partially reversible transitions, and (iv) general three-state models. The method is validated using calorimetry data for chicken egg lysozyme, mutated Protein A, three wild-types of haloalkane dehalogenases, and a mutant stabilized by protein engineering. We show that modelling of reheating increases the precision of determination of unfolding mechanisms, free energies, temperatures, and heat capacity differences. Moreover, this modelling indicates whether alternative refolding pathways might occur upon cooling. The Matlab-based data fitting software tool and its user guide are provided as a supplement.

• MeSH
• Calorimetry methods   MeSH
• Kinetics MeSH
• Chick Embryo MeSH
• Muramidase chemistry   metabolism   MeSH
• Protein Engineering MeSH
• Protein Unfolding MeSH
• Animals MeSH
• Check Tag
• Chick Embryo MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Muramidase MeSH

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 guided through the processes of selecting hot spots using four different protein engineering strategies and optimizing the resulting library's size by narrowing down a set of substitutions at individual randomized positions. The only required input is a query protein structure. The results of the calculations are mapped onto the protein's structure and visualized with a JSmol applet. HotSpot Wizard lists annotated residues suitable for mutagenesis and can automatically design appropriate codons for each implemented strategy. Overall, HotSpot Wizard provides comprehensive annotations of protein structures and assists protein engineers with the rational design of site-specific mutations and focused libraries. It is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard.

• MeSH
• Automation MeSH
• Biocatalysis MeSH
• Databases, Protein MeSH
• Internet * MeSH
• Evolution, Molecular MeSH
• Models, Molecular MeSH
• Mutation * MeSH
• Mutagenesis, Site-Directed methods   MeSH
• Peptide Library * MeSH
• Proteins chemistry   genetics   MeSH
• Software * MeSH
• Protein Stability MeSH
• Amino Acid Substitution MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Peptide Library * MeSH
• Proteins MeSH