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 Pyrococcus horikoshii 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.IMPORTANCE 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.