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.

• Klíčová slova
• DmmarA, Mycobacterium marinum, SAXS, X-ray crystallography, enantioselectivity, haloalkane dehalogenases, homodimerization, surface loops,
• MeSH
• hydrolasy chemie   MeSH
• kyselina aspartová MeSH
• Mycobacterium marinum * metabolismus   MeSH
• stereoizomerie MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• kyselina aspartová 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.

• Klíčová slova
• DhmeA, Haloferax mediterranei, catalysis, cryo-EM, haloalkane dehalogenase, multimerization, x-ray crystallography,
• MeSH
• elektronová kryomikroskopie metody   MeSH
• hydrolasy * chemie   MeSH
• krystalografie rentgenová MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy * 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.

• Klíčová slova
• (S)-enantiopreference, catalytic residues, dehalogenase, enantioselectivity, halide-stabilizing residues, haloalkane, haloalkane dehalogenase, kinetics, mutagenesis, structure, substrate specificity, thermophilic bacterium, thermostability,
• MeSH
• Actinobacteria chemie   genetika   metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• katalýza MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH

A haloalkane dehalogenase, DpcA, from Psychrobacter cryohalolentis K5, representing a novel psychrophilic member of the haloalkane dehalogenase family, was identified and biochemically characterized. DpcA exhibited a unique temperature profile with exceptionally high activities at low temperatures. The psychrophilic properties of DpcA make this enzyme promising for various environmental applications.

• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• fyziologická adaptace * MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• nízká teplota * MeSH
• Psychrobacter enzymologie   genetika   růst a vývoj   fyziologie   MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH

We report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58. DatA possesses a peculiar pair of halide-stabilizing residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity toward racemic mixtures of chiral brominated alkanes and esters.

• MeSH
• Agrobacterium tumefaciens enzymologie   genetika   metabolismus   MeSH
• alkany metabolismus   MeSH
• DNA bakterií chemie   genetika   MeSH
• estery metabolismus   MeSH
• hydrolasy genetika   izolace a purifikace   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• rostliny mikrobiologie   MeSH
• sekvenční analýza DNA MeSH
• stereoizomerie MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkany MeSH
• DNA bakterií MeSH
• estery MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH