8OOH

Cryo-EM map of the focused refinement of the subfamily III haloalkane dehalogenase from Haloferax mediterranei dimer forming hexameric assembly.

Summary for 8OOH

• Entry DOI: 10.2210/pdb8ooh/pdb
• EMDB information: 17015
• Descriptor: Alpha/beta fold hydrolase (1 entity in total)
• Functional Keywords: haloalkane dehalogenase, hydrolase
• Biological source: Haloferax mediterranei
• Total number of polymer chains: 2
• Total formula weight: 71423.59

Authors

Polak, M.,Novacek, J.,Chmelova, K.,Marek, M. (deposition date: 2023-04-05, release date: 2023-10-11)

Primary citation

Chmelova, K.,Gao, T.,Polak, M.,Schenkmayerova, A.,Croll, T.I.,Shaikh, T.R.,Skarupova, J.,Chaloupkova, R.,Diederichs, K.,Read, R.J.,Damborsky, J.,Novacek, J.,Marek, M.
Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and x-ray crystallography.
Protein Sci., 32:e4751-e4751, 2023

PubMed Abstract: Haloalkane dehalogenase (HLD) enzymes employ an S 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 (DhmeA ) that provided diffraction-quality crystals. The 3.3 Å crystal structure reveals that DhmeA 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.

PubMed: 37574754
DOI: 10.1002/pro.4751

Experimental method

ELECTRON MICROSCOPY (7 Å)