Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and x-ray crystallography
|Článek v odborném periodiku
|Časopis / Zdroj
|Fakulta / Pracoviště MU
|catalysis; cryo-EM; DhmeA; haloalkane dehalogenase; Haloferax mediterranei; multimerization; x-ray crystallography
|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.