8CKP

X-ray structure of the crystallization-prone form of subfamily III haloalkane dehalogenase DhmeA from Haloferax mediterranei

Summary for 8CKP

• Entry DOI: 10.2210/pdb8ckp/pdb
• Descriptor: Alpha/beta fold hydrolase, CHLORIDE ION (3 entities in total)
• Functional Keywords: haloalkane dehalogenase-like, hydrolase, unknown function
• Biological source: Haloferax mediterranei
• Total number of polymer chains: 10
• Total formula weight: 357002.52

Authors

Marek, M.,Chmelova, K.,Schenkmayerova, A.,Croll, T.,Read, R.J.,Diederichs, K. (deposition date: 2023-02-16, release date: 2023-08-30, Last modification 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

X-RAY DIFFRACTION (3.31 Å)