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

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Publikace nespadá pod Filozofickou fakultu, ale pod Přírodovědeckou fakultu. Oficiální stránka publikace je na webu muni.cz.
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CHMELOVÁ Klaudia GAO Tadeja POLÁK Martin SMITH Andrea CROLL Tristan I. SHAIKH Tanvir ŠKAŘUPOVÁ Jana CHALOUPKOVÁ Radka DIEDERICHS Kay READ Randy J. DAMBORSKÝ Jiří NOVÁČEK Jiří MAREK Martin

Rok publikování 2023
Druh Článek v odborném periodiku
Časopis / Zdroj Protein Science
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
www https://onlinelibrary.wiley.com/doi/10.1002/pro.4751
Doi http://dx.doi.org/10.1002/pro.4751
Klíčová slova catalysis; cryo-EM; DhmeA; haloalkane dehalogenase; Haloferax mediterranei; multimerization; x-ray crystallography
Přiložené soubory
Popis 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 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(Delta GG)) that provided diffraction-quality crystals. The 3.3 angstrom crystal structure reveals that DhmeA.GG forms a ring-like 20-mer structure with outer and inner diameter of similar to 200 and similar to 80 angstrom, 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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