Characterization of a Glycyl-Specific TET Aminopeptidase Complex from Pyrococcus horikoshii

Abstract

The TET peptidases are large self-compartmentalized complexes that form dodecameric particles. These metallopeptidases, members of the M42 family, are widely distributed in prokaryotes. Three different versions of TET complexes, with different substrate specificities, were found to coexist in the cytosol of the hyperthermophilic archaeon In the present work, we identified a novel type of TET complex that we named PhTET4. The recombinant PhTET4 enzyme was found to self-assemble as a tetrahedral edifice similar to other TET complexes. We determined PhTET4 substrate specificity using a broad range of monoacyl chromogenic and fluorogenic compounds. High-performance liquid chromatographic peptide degradation assays were also performed. These experiments demonstrated that PhTET4 is a strict glycyl aminopeptidase, devoid of amidolytic activity toward other types of amino acids. The catalytic efficiency of PhTET4 was studied under various conditions. The protein was found to be a hyperthermophilic alkaline aminopeptidase. Interestingly, unlike other peptidases from the same family, it was activated only by nickel ions. We describe here the first known peptidase displaying exclusive activity toward N-terminal glycine residues. This work indicates a specific role for intracellular glycyl peptidases in deep sea hyperthermophilic archaeal metabolism. These observations also provide critical evidence for the use of these archaeal extremozymes for biotechnological applications.

How metal cofactors drive dimer–dodecamer transition of the M42 aminopeptidase TmPep1050 of Thermotoga maritima

“…They adopt a peculiar tetrahedron-shaped structure compartmenting the active sites in a buried catalytic chamber (15). In Pyrococcus horikoshii, four TET-aminopeptidases -PhTET1, PhTET2, PhTET3, and PhTET4 -have been described and each of them displays a different substrate specificityaspartyl-, leucyl-, lysyl-, and glycyl-aminopeptidase activity, respectively (15)(16)(17)(18). Remarkably, heterocomplexes, made of PhTET2 and PhTET3, have been reported, leading to the assumption of the peptidasome particle existence (19,20).…”

M42 aminopeptidase catalytic site: the structural and functional role of a strictly conserved aspartate residue

“…1,2 In Pyrococcus horikoshii, four M42 aminopeptidases (PhTET1, PhTET2, PhTET3, and PhTET4) have been described, each having a different, but complementary, substrate specificity. [3][4][5][6] PhTET2 and PhTET3 have even been shown to form heterocomplexes, suggesting that peptidasome particles may exist. 7,8 The M42 aminopeptidases are characterized by a genuine quaternary structure made of twelve subunits organized spatially as a tetrahedron.…”

“…7,8 The M42 aminopeptidases are characterized by a genuine quaternary structure made of twelve subunits organized spatially as a tetrahedron. 1,[3][4][5][6][9][10][11][12][13] The association of the twelve subunits is often described as the assembly of six dimers, each dimer being located at a tetrahedron edge. 1 Four gates are found at the middle of the tetrahedron faces, leading to a wide inner chamber.…”

“…The M42 aminopeptidases are dinuclear enzymes proposed to achieve peptide degradation downstream the proteasome in Bacteria and Archaea 1,2 . In Pyrococcus horikoshii , four M42 aminopeptidases (PhTET1, PhTET2, PhTET3, and PhTET4) have been described, each having a different, but complementary, substrate specificity 3‐6 . PhTET2 and PhTET3 have even been shown to form heterocomplexes, suggesting that peptidasome particles may exist 7,8 .…”

“…PhTET2 and PhTET3 have even been shown to form heterocomplexes, suggesting that peptidasome particles may exist 7,8 . The M42 aminopeptidases are characterized by a genuine quaternary structure made of twelve subunits organized spatially as a tetrahedron 1,3‐6,9‐13 . The association of the twelve subunits is often described as the assembly of six dimers, each dimer being located at a tetrahedron edge 1 .…”