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 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