Determining why convergent traits use distinct versus shared genetic components is crucial for understanding how evolutionary processes generate and sustain biodiversity. However, the factors dictating the genetic underpinnings of convergent traits remain incompletely understood. Here, we use heterologous protein expression, biochemical assays, and phylogenetic analyses to confirm the origin of a luciferase gene from haloalkane dehalogenases in the brittle star Amphiura filiformis. Through database searches and gene tree analyses, we also show a complex pattern of the presence and absence of haloalkane dehalogenases across organismal genomes. These results first confirm parallel evolution across a vast phylogenetic distance, because octocorals like Renilla also use luciferase derived from haloalkane dehalogenases. This parallel evolution is surprising, even though previously hypothesized, because many organisms that also use coelenterazine as the bioluminescence substrate evolved completely distinct luciferases. The inability to detect haloalkane dehalogenases in the genomes of several bioluminescent groups suggests that the distribution of this gene family influences its recruitment as a luciferase. Together, our findings highlight how biochemical function and genomic availability help determine whether distinct or shared genetic components are used during the convergent evolution of traits like bioluminescence.

• Keywords
• bioluminescence, convergent evolution, haloalkane dehalogenase, luciferase, parallel evolution,
• MeSH
• Biocatalysis MeSH
• Echinodermata * genetics   metabolism   MeSH
• Phylogeny MeSH
• Genome * MeSH
• Genomics MeSH
• Hydrolases * genetics   metabolism   MeSH
• Kinetics MeSH
• Luciferases * genetics   metabolism   MeSH
• Evolution, Molecular * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• haloalkane dehalogenase MeSH Browser
• Hydrolases * MeSH
• Luciferases * MeSH

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 to understand their catalytic and noncatalytic functions. Here, crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85 Å resolution. The structures show a canonical αβα-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate-histidine-aspartate) and uncommon active-site pocket reveal the molecular specificities of a catalytic apparatus that exhibits a rare (S)-enantiopreference. Additionally, the structures reveal a previously unobserved mode of symmetric homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering. Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its uncommon enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution.

• Keywords
• DmmarA, Mycobacterium marinum, SAXS, X-ray crystallography, enantioselectivity, haloalkane dehalogenases, homodimerization, surface loops,
• MeSH
• Hydrolases chemistry   MeSH
• Aspartic Acid MeSH
• Mycobacterium marinum * metabolism   MeSH
• Stereoisomerism MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH
• Aspartic Acid MeSH

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

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 reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.

• Keywords
• ancestral sequence reconstruction, haloalkane dehalogenase, halogenated pollutants, structural analysis,
• MeSH
• Hydrolases chemistry   metabolism   MeSH
• Hydrolysis MeSH
• Catalytic Domain MeSH
• Crystallography, X-Ray methods   MeSH
• Evolution, Molecular MeSH
• Models, Molecular MeSH
• Mycobacterium bovis enzymology   MeSH
• Protein Engineering methods   MeSH
• Sequence Analysis, Protein methods   MeSH
• Sphingomonadaceae enzymology   MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH

Haloalkane dehalogenases can cleave a carbon-halogen bond in a broad range of halogenated aliphatic compounds. However, a highly conserved catalytic pentad composed of a nucleophile, a catalytic base, a catalytic acid, and two halide-stabilizing residues is required for their catalytic activity. Only a few family members, e.g., DsaA, DmxA, or DmrB, remain catalytically active while employing a single halide-stabilizing residue. Here, we describe a novel haloalkane dehalogenase, DsvA, from a mildly thermophilic bacterium, Saccharomonospora viridis strain DSM 43017, possessing one canonical halide-stabilizing tryptophan (W125). At the position of the second halide-stabilizing residue, DsvA contains the phenylalanine F165, which cannot stabilize the halogen anion released during the enzymatic reaction by a hydrogen bond. Based on the sequence and structural alignments, we identified a putative second halide-stabilizing tryptophan (W162) located on the same α-helix as F165, but on the opposite side of the active site. The potential involvement of this residue in DsvA catalysis was investigated by the construction and biochemical characterization of the three variants, DsvA01 (F165W), DsvA02 (W162F), and DsvA03 (W162F and F165W). Interestingly, DsvA exhibits a preference for the (S)- over the (R)-enantiomers of β-bromoalkanes, which has not been reported before for any characterized haloalkane dehalogenase. Moreover, DsvA shows remarkable operational stability at elevated temperatures. The present study illustrates that protein sequences possessing an unconventional composition of catalytic residues represent a valuable source of novel biocatalysts.IMPORTANCE The present study describes a novel haloalkane dehalogenase, DsvA, originating from a mildly thermophilic bacterium, Saccharomonospora viridis strain DSM 43017. We report its high thermostability, remarkable operational stability at high temperatures, and an (S)-enantiopreference, which makes this enzyme an attractive biocatalyst for practical applications. Sequence analysis revealed that DsvA possesses an unusual composition of halide-stabilizing tryptophan residues in its active site. We constructed and biochemically characterized two single point mutants and one double point mutant and identified the noncanonical halide-stabilizing residue. Our study underlines the importance of searching for noncanonical catalytic residues in protein sequences.

• Keywords
• (S)-enantiopreference, catalytic residues, dehalogenase, enantioselectivity, halide-stabilizing residues, haloalkane, haloalkane dehalogenase, kinetics, mutagenesis, structure, substrate specificity, thermophilic bacterium, thermostability,
• MeSH
• Actinobacteria chemistry   genetics   metabolism   MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Hydrolases chemistry   genetics   metabolism   MeSH
• Catalysis MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH

Ancestral sequence reconstruction is a powerful method for inferring ancestors of modern enzymes and for studying structure-function relationships of enzymes. We have previously applied this approach to haloalkane dehalogenases (HLDs) from the subfamily HLD-II and obtained thermodynamically highly stabilized enzymes (ΔT m up to 24 °C), showing improved catalytic properties. Here we combined crystallographic structural analysis and computational molecular dynamics simulations to gain insight into the mechanisms by which ancestral HLDs became more robust enzymes with novel catalytic properties. Reconstructed ancestors exhibited similar structure topology as their descendants with the exception of a few loop deviations. Strikingly, molecular dynamics simulations revealed restricted conformational dynamics of ancestral enzymes, which prefer a single state, in contrast to modern enzymes adopting two different conformational states. The restricted dynamics can potentially be linked to their exceptional stabilization. The study provides molecular insights into protein stabilization due to ancestral sequence reconstruction, which is becoming a widely used approach for obtaining robust protein catalysts.

• Keywords
• Ancestral sequence reconstruction, Conformational flexibility, Enzyme, Haloalkane dehalogenase, Protein design, Protein simulations, Thermostability, X-ray crystallography,
• Publication type
• Journal Article MeSH

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 biochemically characterized wild type haloalkane dehalogenases belonging to subfamily II. The enzyme was successfully expressed and its crystal structure was solved at 1.45 Å resolution. DmxA structure contains several features distinct from known members of haloalkane dehalogenase family: (i) a unique composition of catalytic residues; (ii) a dimeric state mediated by a disulfide bridge; and (iii) narrow tunnels connecting the enzyme active site with the surrounding solvent. The importance of narrow tunnels in such paradoxically high stability of DmxA enzyme was confirmed by computational protein design and mutagenesis experiments.

• Keywords
• access tunnel, catalytic pentad, dimer, enantiselectivity, haloalkane dehalogenase, psychrophile, thermostability,
• Publication type
• Journal Article MeSH

Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.

• Keywords
• Psychrobacter cryohalolentis, X-ray diffraction, haloalkane dehalogenase, psychrophiles, structural analysis, α/β-hydrolase,
• MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Gene Expression MeSH
• Genetic Vectors chemistry   metabolism   MeSH
• Hydrocarbons, Halogenated chemistry   metabolism   MeSH
• Hydrolases chemistry   genetics   metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Cloning, Molecular MeSH
• Protein Conformation, alpha-Helical MeSH
• Protein Conformation, beta-Strand MeSH
• Crystallography, X-Ray MeSH
• Cold Temperature MeSH
• Psychrobacter chemistry   enzymology   MeSH
• Recombinant Fusion Proteins chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Molecular Docking Simulation MeSH
• Structural Homology, Protein MeSH
• Substrate Specificity MeSH
• Thermodynamics MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 1-bromohexane MeSH Browser
• Bacterial Proteins MeSH
• haloalkane dehalogenase MeSH Browser
• Hydrocarbons, Halogenated MeSH
• Hydrolases MeSH
• Recombinant Fusion Proteins MeSH

We emphasize the importance of dynamics and hydration for enzymatic catalysis and protein design by transplanting the active site from a haloalkane dehalogenase with high enantioselectivity to nonselective dehalogenase. Protein crystallography confirms that the active site geometry of the redesigned dehalogenase matches that of the target, but its enantioselectivity remains low. Time-dependent fluorescence shifts and computer simulations revealed that dynamics and hydration at the tunnel mouth differ substantially between the redesigned and target dehalogenase.

• MeSH
• Hydrocarbons, Brominated chemistry   MeSH
• Spectrometry, Fluorescence MeSH
• Hydrolases chemistry   genetics   MeSH
• Catalytic Domain MeSH
• Catalysis MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Molecular Sequence Data MeSH
• Mutagenesis, Site-Directed MeSH
• Protein Engineering * MeSH
• Amino Acid Sequence MeSH
• Molecular Dynamics Simulation * MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
• Stereoisomerism MeSH
• Water chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hydrocarbons, Brominated MeSH
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH
• Water MeSH

The effect of non-denaturing concentrations of three different organic solvents, formamide, acetone and isopropanol, on the structure of haloalkane dehalogenases DhaA, LinB, and DbjA at the protein-solvent interface was studied using molecular dynamics simulations. Analysis of B-factors revealed that the presence of a given organic solvent mainly affects the dynamical behavior of the specificity-determining cap domain, with the exception of DbjA in acetone. Orientation of organic solvent molecules on the protein surface during the simulations was clearly dependent on their interaction with hydrophobic or hydrophilic surface patches, and the simulations suggest that the behavior of studied organic solvents in the vicinity of hyrophobic patches on the surface is similar to the air/water interface. DbjA was the only dimeric enzyme among studied haloalkane dehalogenases and provided an opportunity to explore effects of organic solvents on the quaternary structure. Penetration and trapping of organic solvents in the network of interactions between both monomers depends on the physico-chemical properties of the organic solvents. Consequently, both monomers of this enzyme oscillate differently in different organic solvents. With the exception of LinB in acetone, the structures of studied enzymes were stabilized in water-miscible organic solvents.

• MeSH
• 2-Propanol chemistry   pharmacology   MeSH
• Acetone chemistry   pharmacology   MeSH
• Formamides chemistry   pharmacology   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Hydrolases chemistry   MeSH
• Crystallography, X-Ray MeSH
• Protein Structure, Quaternary drug effects   MeSH
• Models, Molecular MeSH
• Solvents chemistry   MeSH
• Molecular Dynamics Simulation MeSH
• Protein Structure, Tertiary drug effects   MeSH
• Water chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2-Propanol MeSH
• Acetone MeSH
• formamide MeSH Browser
• Formamides MeSH
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH
• Solvents MeSH
• Water MeSH