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Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and X-ray crystallography

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2023

Autor:innen

Chmelova, Klaudia
Gao, Tadeja
Polak, Martin
Schenkmayerova, Andrea
Croll, Tristan I.
Shaikh, Tanvir R.
Skarupova, Jana
Chaloupkova, Radka
Marek, Martin
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Protein Science. Wiley. 2023, 32(10), e4751. ISSN 0961-8368. eISSN 1469-896X. Available under: doi: 10.1002/pro.4751

Zusammenfassung

Haloalkane dehalogenase (HLD) enzymes employ an SN2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but a 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 Å 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.

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570 Biowissenschaften, Biologie

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Molecular Biology

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ISO 690CHMELOVA, Klaudia, Tadeja GAO, Martin POLAK, Andrea SCHENKMAYEROVA, Tristan I. CROLL, Tanvir R. SHAIKH, Jana SKARUPOVA, Radka CHALOUPKOVA, Kay DIEDERICHS, Martin MAREK, 2023. Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and X-ray crystallography. In: Protein Science. Wiley. 2023, 32(10), e4751. ISSN 0961-8368. eISSN 1469-896X. Available under: doi: 10.1002/pro.4751
BibTex
@article{Chmelova2023-09-15Multi-67703,
  year={2023},
  doi={10.1002/pro.4751},
  title={Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and X-ray crystallography},
  number={10},
  volume={32},
  issn={0961-8368},
  journal={Protein Science},
  author={Chmelova, Klaudia and Gao, Tadeja and Polak, Martin and Schenkmayerova, Andrea and Croll, Tristan I. and Shaikh, Tanvir R. and Skarupova, Jana and Chaloupkova, Radka and Diederichs, Kay and Marek, Martin},
  note={Article Number: e4751}
}
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