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 are hydrolytic enzymes with a broad range of potential practical applications such as biodegradation, biosensing, biocatalysis and cellular imaging. Two newly isolated psychrophilic haloalkane dehalogenases exhibiting interesting catalytic properties, DpcA from Psychrobacter cryohalolentis K5 and DmxA from Marinobacter sp. ELB17, were purified and used for crystallization experiments. After the optimization of crystallization conditions, crystals of diffraction quality were obtained. Diffraction data sets were collected for native enzymes and complexes with selected ligands such as 1-bromohexane and 1,2-dichloroethane to resolutions ranging from 1.05 to 2.49 Å.

• Keywords
• DmxA, DpcA, Marinobacter sp. ELB17, Psychrobacter cryohalolentis K5, haloalkane dehalogenases,
• MeSH
• Bacterial Proteins analysis   chemistry   MeSH
• X-Ray Diffraction MeSH
• Hydrolases analysis   chemistry   MeSH
• Catalytic Domain MeSH
• Crystallography, X-Ray MeSH
• Marinobacter enzymology   MeSH
• Psychrobacter enzymology   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

Haloalkane dehalogenases hydrolyze carbon-halogen bonds in a wide range of halogenated aliphatic compounds. The potential use of haloalkane dehalogenases in bioremediation applications has stimulated intensive investigation of these enzymes and their engineering. The mutant DhaA31 was constructed to degrade the anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. This strategy enhances activity towards TCP by decreasing the accessibility of the active site to water molecules, thereby promoting formation of the activated complex. The structure of DhaA31 will help in understanding the structure-function relationships involved in the improved dehalogenation of TCP. The mutant protein DhaA31 was crystallized by the sitting-drop vapour-diffusion technique and crystals of DhaA31 in complex with TCP were obtained using soaking experiments. Both crystals belonged to the triclinic space group P1. Diffraction data were collected to high resolution: to 1.31 Å for DhaA31 and to 1.26 Å for DhaA31 complexed with TCP.

• MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• X-Ray Diffraction MeSH
• Hydrolases chemistry   genetics   metabolism   MeSH
• Crystallization MeSH
• Molecular Sequence Data MeSH
• Propane analogs & derivatives   chemistry   metabolism   MeSH
• Rhodococcus enzymology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1,2,3-trichloropropane MeSH Browser
• Bacterial Proteins MeSH
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH
• Propane MeSH

Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon-halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2(1)2(1)2(1) as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively.

• MeSH
• 2-Propanol MeSH
• Bacterial Proteins chemistry   MeSH
• X-Ray Diffraction MeSH
• Hydrolases chemistry   genetics   metabolism   MeSH
• Hydrolysis MeSH
• Isoenzymes chemistry   genetics   MeSH
• Catalysis MeSH
• Crystallization MeSH
• Crystallography, X-Ray methods   MeSH
• Ligands MeSH
• Propane analogs & derivatives   MeSH
• Rhodococcus enzymology   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• 1,2,3-trichloropropane MeSH Browser
• 2-Propanol MeSH
• Bacterial Proteins MeSH
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH
• Isoenzymes MeSH
• Ligands MeSH
• Propane MeSH