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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00159816%3A_____%2F23%3A00079716" target="_blank" >RIV/00159816:_____/23:00079716 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216224:14310/23:00132287

Výsledek na webu

<a href="https://onlinelibrary.wiley.com/doi/10.1002/pro.4751" target="_blank" >https://onlinelibrary.wiley.com/doi/10.1002/pro.4751</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1002/pro.4751" target="_blank" >10.1002/pro.4751</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2023

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

PROTEIN SCIENCE

ISSN

0961-8368

e-ISSN

1469-896X

Svazek periodika

32

Číslo periodika v rámci svazku

10

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

22

Strana od-do

"e4751"

Kód UT WoS článku

001067173900001

EID výsledku v databázi Scopus

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