Etudes structurales et propriétés enzymatiques de deux nouvelles aminopeptidases TETs auto-compartimentées chez les archées

Abstract : Aminopeptidases represent a group of enzymes displaying key cellular function inphysiological and pathological mechanisms. They are involved in the enzymatic cascade beyond the action of endoproteases, in homeostasis through the renewal of the amino acid pool, in the energy metabolism, in the regulation of bioactive peptide activities, in the antigen presentation and in a diversity of pathological mechanisms such as neurological diseases as well as viral and parasitic infections. Aminopeptidases TET are able of forming tetrahedral macro-assemblies built by twelve subunits. In order to better understand their biological function and their mode of action, we studied the functional and structural properties of two novel TET complexes derived from hyperthermophilic archaea. The hyperthermophilic archaeon Methanocaldococcus jannaschii has only one version of TET (MjTET) that was produced in Escherichia coli and purified as dodecameric macromolecule. The search for its enzymatic activity and peptide substrates by using chromogenic/fluorogenic assays and reverse phase HPLC studies, demonstrated that this enzyme is a cobalt-activated leucine aminopeptidase, discriminated from other M42 aminopeptidases by its very broad activity spectrum, that extends to aromatic residues. Complete structure of this aminopeptidase was determined by combining X-ray crystallography (2.4 Å) and cryo-electron microscopy (4.1 Å). Analysis of MjTET specificity pocket indicated possible molecular bases for substrate discrimination in TET peptidases. In depth investigation of the particle internal structure allowed to propose a novel peptide trafficking mechanism for the TET family tetrahedral particles. Three types of TET complexes are present in the hyperthermophilic archaea, Pyrococcus horikoshii. The study of an unassigned protein displaying ~20% identity with the PhTETs systems allowed us to identify a fourth version of TET complex in this organism: PhTET4. The recombinant protein was purified. It formed tetrahedral dodecameric complex. Biochemical studies indicated that the enzyme has a very narrow hydrolytic specificity directed exclusively toward the peptide N-terminal glycine residues. In addition, this enzyme is activated by nickel ions. These features allowed proposing that, in archaea, the multiplicity of specialized TET systems could be associated with heterotrophy while unique TET system displaying “housekeeping” function is present in autotrophic organisms.
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Hind Basbous. Etudes structurales et propriétés enzymatiques de deux nouvelles aminopeptidases TETs auto-compartimentées chez les archées. Biologie structurale [q-bio.BM]. Université Grenoble Alpes, 2016. Français. ⟨NNT : 2016GREAV016⟩. ⟨tel-01842280⟩

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