Patent Description:
During the last decade, it has been found that several enzymes from M42 and M18 metallopeptidase families self-assembled in half megadalton complexes made of twelve subunits. These proteins were named TET because of the tetrahedral shape adopted by these dodecameric edifices.

TET complex was first purified from halophilic archaea during a search for protein quality control machines involved in low salt stress. This novel type of cage-forming protease attracted interest since it is present in many cell types and has homologues in all kingdoms of life. Previous structural studies revealed that all TET dodecamers share similar architectures: the twelve monomers are associated in a tetrahedral particle with four large substrate entry pores formed by the junction of six subunits situated on each facet and four catalytic chambers each comprising three active sites of three monomers which formed the apex of tetrahedron. This architecture is strikingly different from the barrel-shaped one adopted by other cytosolic compartmentalized peptidases. TET assembling mechanism from dimeric precursors was determined using a combination of biophysical techniques. They demonstrated that it is a highly ordered process involving predominantly hexameric precursors.

Biochemical characterizations of TET complex in vivo and in vitro indicated that the purpose of self-compartmentalization in the M42 peptidases is to trigger their amidolytic activity toward long peptides. Different types of TET enzymes were found in archaea, bacteria and eukarya and different versions of enzymatic complexes often co-exist in the cytosol of the same organism.

All M42 TET complexes characterized so far are cobalt-activated aminopeptidases able to degrade oligopeptides to single amino acids (<NPL>). However, enzymatic studies revealed that they displayed marked substrate preferences. This aspect of TET biochemistry was mostly studied in Pyrococcus horikoshii, a deep-sea hyperthermophilic Euryarchaeota that contains three different versions of TET complex: PhTET1, a glutamyl-aspartyl aminopeptidase, PhTET2, a leucyl aminopeptidase with broad activity against neutral amino acids, and PhTET3, a lysyl aminopeptidase.

In these complexes, the comparison of the "S1" binding pocket properties supports a clear correlation between charge distribution and substrate specificities.

Despite important progress in determining the structure-function relationship of TET machinery, many fundamental questions remain about the cellular function and the mode of action of TET system. In particular, it is not fully understood the detailed mechanism of polypeptide substrate filtering and guidance to the active site as well as the amino acid-product expulsion system remains elusive.

A genomic survey on the occurrence of TET-like enzymes in prokaryotic genomes revealed that some archaea possess up to four versions of TET proteins while others only express one version. In order to specify the basic physiological roles of TET system and the reasons underlying the multiplicity of TET cellular machines in some microorganisms and to bring further insights into their structure-function relationship, the inventors studied the biochemical and structural properties of the unique TET complex from Methanocaldococcus jannaschii, a deep-sea hyperthermophilic archaea. So there is a need to provide new peptidases for industry.

The invention relates to the use of a TET protein as a N-terminus aromatic amino acid residues specific exopeptidase, said TET protein consisting of the amino acid sequence as set forth in SEQ ID NO: <NUM>,
or any homologous protein derived from said TET protein as set forth in SEQ ID NO: <NUM> by substitution, addition or deletion of at least one amino acid, provided that the derived protein retains at least <NUM> % of identity with the amino acid sequence as set forth in SEQ ID NO: <NUM>, and said derived protein retaining a N-terminus aromatic amino acid residues specific exopeptidase activity.

The invention is based on the characterization by the inventors of the enzymatic activity of MjTET. This characterization revealed that this archaeal TET aminopeptidase displayed broad substrate specificity, thus indicating a housekeeping function devoted to the non-specific destruction of polypeptides. More advantageously, the inventors identified that MjTET is a TET protein having a specificity toward the N-terminus aromatic amino acids, said activity vis-à-vis aromatic residues being largely enhanced compared to the other TET protein that where characterized to date.

Moreover, the inventors also identify that the protein according to the invention harbors an aminopeptidase activity toward all the amino acids except aspartic acid, cysteine and proline. This means that if these amino acids are present in the N-terminus of a peptide, a polypeptide or a protein, the protein according to the invention will not be able to brake the peptidic bound.

Advantageously, in another aspect, the invention relates to the use of a TET protein as a N-terminus exopeptidase provided that the amino acid at the N-terminus is not aspartic acid, cystein or proline, said TET protein consisting of the amino acid sequence as set forth in SEQ ID NO: <NUM>,
or any homologous protein derived from said TET protein as set forth in SEQ ID NO: <NUM> by substitution, addition or deletion of at least one amino acid, provided that the derived protein retains at least <NUM> % of identity with the amino acid sequence as set forth in SEQ ID NO: <NUM>, and said derived protein retaining a N-terminus specific exopeptidase activity.

The inventors were also able to identify the structure of the protein. Structural analysis combining X-ray crystallography and cryo-electron microscopy allowed providing complete MjTET atomic model. In this model, internal network of mobile loops that were poorly defined in previous crystallographic studies could be observed. The topological analysis of these loops highlights an elegant peptide guiding system toward catalytic sites.

In the invention the protein comprising the amino acid sequence SEQ ID NO : <NUM> is called MjTET.

The invention also relates to the use of at least a TET protein harboring at least a N-terminus aromatic amino acid residues specific exopeptidase activity, for the modification of all or part of the polypeptide content of a substrate comprising peptides, polypeptides and/or proteins, said at least a TET protein consisting of the amino acid sequence as set forth in SEQ ID NO: <NUM>,
or any homologous protein derived from said at least a TET protein as set forth in SEQ ID NO: <NUM> by substitution, addition or deletion of at least one amino acid, provided that the derived protein retains at least <NUM> % of identity with the amino acid sequence as set forth in SEQ ID NO: <NUM>, and said derived protein retaining a N-terminus aromatic amino acid residues specific exopeptidase activity.

By "exopeptidase", it is meant in the invention a peptidase that catalyses the cleavage of the terminal peptide bond of an amino acid chain, starting either from the amino or carboxyl terminal of the said amino acid chain.

By "peptide", it is meant in the invention an amino acid chain comprising at least <NUM> amino acids. Peptides can be obtained either from protein degradation or from chemical synthesis. By "polypeptide", it is meant in the invention an amino acid chain larger than a peptide and obtained from degradation of proteins and not from chemical synthesis. Peptides and polypeptides may harbour biological function, but said function is not associated with a cellular process. By "protein", it is meant in the invention an amino acid chain containing molecule harbouring biological function and which is found naturally in an organism, said biological function being part of a natural process of the cell.

By "at least <NUM>% of identity with the sequence as set forth in SEQ ID NO : <NUM>", it is meant in the invention <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> % and <NUM> % of identity with the sequence as set forth in SEQ ID NO : <NUM>. Regarding the percentage of identity, it is defined by the percentage of amino acid residues of SEQ ID NO : <NUM> which align with the same amino acid in the sequence of the homologous protein. The sequence alignment is carried out using dedicated algorithms and programs (such as ClustalW, for instance).

By "modification of all or part of the polypeptide", it is meant in the invention that the modification of a peptide can result in the removal of one or more amino acid from the peptide. If the peptide contains only aromatic amino acids, the peptide can be completely degraded, i.e. can be converted into the free amino acids that constitute the peptide.

Moreover, the "modification of all or part of the polypeptide", means also in the invention that, if a composition contains two or more peptides, at least one peptide will be degraded by contacted the TET protein according to the invention. If only some peptides are degraded, the composition of peptide will be considered to be partially modified. If all the peptides are subjected to a degradation, the composition of peptide will be considered to be totally modified.

In the invention, regarding a peptide, a polypeptide, a protein or a polypeptide content, the terms "modification" and "degradation" can be used uniformly. In the invention, the term "comprising" is meant to include the terms "consisting essentially of" and "consisting of".

The aromatic amino acids encompassed by the scope of the invention are phenylalanine (Phe; F), tryptophan (Trp; W), tyrosine (Tyr; Y) and histidine (His; H). Thus according to the invention, if a peptide, a polypeptide or a protein contain in its amino-terminal part one amino acid chosen among F, W, Y and H, said amino acid could be released from the peptide by contacting the TET protein, according to the invention, i.e. the MjTET protein.

Advantageously, the invention relates to the use as defined above, wherein said TET protein or said derived protein originates from an extremophile microorganism belonging to the Methanococcales order and are isolated from this extremophile microorganism.

By "extremophile", it is meant in the invention an organism that thrives in physically or geochemically extreme conditions that are detrimental to most life on Earth. In contrast, organisms that live in more moderate environments may be termed mesophiles or neutrophiles. The order Methanococcales consists of <NUM> genera, Methanocaldococcus (hyperthermophiles), Methanotorris (extreme thermophiles), Methanothermococcus (moderate thermophiles) and Methanococcus (mesophiles).

The Methanococcales are strictly oxygen-intolerant and obtain energy by reducing CO<NUM> with H<NUM> to generate methane. They can also scavenge small organic molecules. They move with two bundles of flagella.

The first species of Methanococcales to be isolated came from a sediment sample collected from the sea floor. The sample was found at the base of a <NUM>-foot deep "white smoker" chimney located on the east Pacific Rise.

Advantageously, the invention relates to the use as defined above, wherein said extremophile microorganism is Methanocaldocuccus jannaschii.

Methanococcus jannaschii is an autotropic hyperthermophillic organism that belongs to the kingdom of Archaea. They were found to live in extreme environments such as hydrothermal vents at the bottom of the oceans in which water reaches boiling temperature or pressure is extremely high.

Advantageously, the invention relates to the use as defined above, wherein peptides, polypeptides and/or proteins of said substrate are obtained from food, chemical and health industries, or from natural biomass.

The generation of protein-rich industrial wastes is very high (only from sunflower, about one million tons in Spain). These wastes are not used at all, or are underused in the form of low added-value products. This type of by-products constitutes a reservoir of proteins with a great economic potential.

The TET protein according to the invention, in view of its activity, can be used in various domains for instance, but without limitation:.

The invention also relates to a method for degrading, from the N-terminus part, a polypeptide harboring an aromatic residue at its N-terminal part, said method comprising a step of contacting said polypeptide harboring an aromatic residue at its N-terminal part with.

and possibly a step of recovering the resulting N-terminal aromatic amino acid residue free peptides.

According to the invention, in order to carry out a degradation of a peptide, a polypeptide or a protein, which contains aromatic amino acids in its N-terminus part, the TET protein or the homologous protein of said TET protein is contacted with the peptide to be degraded. The TET protein and the substrate are incubated in an appropriated buffer, as disclosed in the Example.

It is advantageous, when the resulting peptide, polypeptide or protein has been degraded, resulting from the action of the TET protein, to recover, or purify said resulting N-terminal aromatic amino acid residue free peptide, polypeptide or protein. From his general knowledge, the skilled person can easily carry out such purification step, for instance by using chromatography methods or immunological methods.

The invention also relates to a method for modifying all or part of the polypeptide content of a substrate comprising peptides, polypeptides and/or proteins, said method comprising a step of contacting said substrate with.

and possibly a step of recovering the modified polypeptide content of a substrate.

Advantageously, the invention relates to the method, or the use, as defined above, wherein said step of contacting is carried out at a pH range varying from <NUM> to <NUM>.

As demonstrated in the examples, the inventors identified that the TET protein according to the invention is stable in a large range of pH and is active on its substrate from acid medium to basic medium. The TET protein according to the invention is active at pH <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>. The optimal pH range is from <NUM>,<NUM> to <NUM>, wherein the activity of the protein varies from <NUM>% to <NUM>% of the optimal activity, measured as the µmol of substrat per mg of MjTET per min.

Advantageously, the invention relates to the method, or the use as defined above, wherein said step of contacting is carried out at a temperature range varying from <NUM> to <NUM>.

As a thermostable protein, the TET protein according to the invention is active, i.e. can catalyse the degradation of a peptide, a polypeptide or a protein, by removing amino terminus aromatic amino acids residues at high temperatures varying from <NUM> to <NUM>. This means that the protein is active at the following temperatures <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> , <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

Advantageously, the invention relates to the method, or the use as defined above, wherein said step of contacting is carried out in presence of cobalt ions.

The inventors also shown that the TET protein according to the invention is more active when Cobalt ions are present as cofactor that activate the enzyme.

Thus, and more advantageously, the invention relates to the method, or the use, as defined above, wherein the step of contacting is carried out :.

Compared to other TET family member peptidases, the TET protein according to the invention is active at a pressure varying from <NUM>,<NUM> MPa to <NUM> MPa.

Advantageously, the invention relates to the above method, wherein said at least a TET protein or said derived protein is immobilized on a solid support, preferably on a filter cartridge, silica, or magnetic beads.

In view of the robustness of the TET protein, and the stability of the structure, it is possible to immobilized the TET protein on a support. Such a support is advantageous and allows to carry out a peptide, polypeptide or peptide degradation and recover the resulting degraded peptide, polypeptides and protein, easily without additional step of separation of the enzyme and the resulting product.

In another advantageous embodiment, the invention relates to the method, or the use as defined above, wherein said at least a TET protein or said derived protein is expressed in a mesophilic host, from a plasmid, and then purified from cell lysates by a thermal denaturation step of the host proteins.

In order to produce the TET protein, and carry the method above defined, it is possible to produce the TET protein in a mesophilic host, for instance by overexpression of the TET protein the nucleic acid sequence of which being contained in a plasmid, or a vector. By culturing the mesophilic host defined above, it is possible to recover, i.e. purify, the TET protein according to the invention by lysis protocols well known in the art. The TET protein can also be easily purified by denaturing the proteins of the host at a temperature where the TET protein remains active, from <NUM> to <NUM>.

The invention also relates to the use of a solid support for the modification of all or part of the polypeptide content of a substrate comprising peptides, polypeptides and/or proteins, wherein is immobilized on said solid support:.

Advantageously, the invention relates to the use as defined above, wherein said solid support is a filter cartridge, silica or magnetic beads.

Advantageously, said solid support is a filter cartridge or silica beads or magnetic beads or on organic polymeric materials, or on inorganic polymeric materials or on membrane devices or in microcapsules. Membrane devices include hollow fibres.

Alternatively, said at least TET protein or said derived protein is immobilized as crosslinked enzyme aggregates (CLEAs).

CLEAs are developed by precipitation of the enzyme from a solution by adding salt, such as ammonium sulphate, or water-miscible organic solvent, followed by crosslinking with a bifunctional reagent (<NPL>.

Advantageously, the invention relates to the use as defined above, wherein peptides, polypeptides and/or proteins of said substrate are obtained from food, chemical and health industries, or from biomass, as above mentioned. Advantageously, peptides, polypeptides and/or proteins of said substrate are obtained from food, chemical and health industries, or from biomass, as mentioned above. Advantageously, proteins from fermented products or soya products or sea food products or cheese products.

The invention will be better understood from the following examples and the <NUM> following figures.

Genes encoding for MjTET and its tagged form MjTET-Nt (with N-terminal His<NUM> tag) were synthesized and cloned by GeneCust Europe in pET-41c and pET28a+ vectors, respectively. The inventors used Escherichia coli ArcticExpress (DE3)-RIL strain (Agilent Technologies) for the over-expression of these two recombinant proteins. The cells were grown in <NUM> of lysogeny broth (LB) medium containing gentamycin and kanamycine at <NUM>µg/ml. Then the cells were incubated with shaking at <NUM> until the A<NUM> reached <NUM>-<NUM>, at this stage the induction was carried out by adding isopropyl β-D-thiogalactopyranoside at final concentration of <NUM> and proteins are overexpressed during <NUM> at <NUM>. The induced cells were harvested by centrifugation, and the pellets were then conserved at -<NUM> until use. These pellets were suspended in <NUM> of <NUM> Tris-HCl, <NUM> NaCl and <NUM>,<NUM> % Triton X-<NUM>, pH <NUM>, supplemented with <NUM> of lysozyme (Euromedex), <NUM> of DNase I grade II, <NUM> of RNase, <NUM> of Pefabloc SC (Roche), <NUM>µl of <NUM> MgSO<NUM> and <NUM> imidazole for cells expressing MjTET-Nt protein. The cell disruption was achieved by sonication in a Branson sonifier at <NUM> watts for <NUM> cycles on/off of <NUM> sec each, at <NUM>. The crude extracts were heated at <NUM> for <NUM> and later clarified by centrifugation (JA20 rotor) at <NUM> rpm for <NUM> at <NUM>. The supernatant was diluted two times with <NUM> Tris-HCl pH <NUM>, and loaded on anion exchange column (Resource Q) (GE Healthcare Life Sciences) for MjTET recombinant form. The protein was eluted with a linear NaCl gradient from <NUM>-<NUM> in <NUM> Tris-HCl pH <NUM>. While MjTET-Nt purification was performed on HiTrap Chelating HP column (GE Healthcare Life Sciences) with <NUM> and <NUM> imidazole in <NUM> Tris-HCl, <NUM> NaCl, pH <NUM>. Exclusion chromatography for the two protein forms was done on Superose <NUM> column or Superdex <NUM> column (GE Healthcare Life Sciences) previously equilibrated with <NUM> Tris-HCl, <NUM> NaCl, pH <NUM>. The fractions containing protein (<NUM>/<NUM> kDa) according to SDS-PAGE were pooled and concentrated using Amicon cell (Millipore) with a molecular mass cutoff of <NUM> kDa and kept at <NUM>. <NUM> and <NUM> of MjTET and MjTET-Nt were produced from <NUM> of culture, respectively.

MjTET samples, at approximately <NUM>/mL in <NUM> Tris-HCl and <NUM> NaCl, pH <NUM>, were adsorbed to the clean side of a carbon film on mica, stained with <NUM> % sodium silicotungstate pH <NUM> and transferred to a <NUM>-mesh copper grid. The images were taken under low dose conditions (<<NUM> e-/Å<NUM>) at a magnification of <NUM> and <NUM> kX times with defocus values between <NUM> and <NUM> on a Tecnai <NUM> LaB6 electron microscope at <NUM> kV accelerating voltage using CCD Camera Gatan Orius <NUM>.

The effect of pH on MjTET activity was determined in pH range varying from <NUM> to <NUM>. The buffers used were: PIPES, pH <NUM>-<NUM>; CHES, pH <NUM>-<NUM> and CAPS, pH <NUM>-<NUM>. All buffers were used at <NUM> concentration in presence of <NUM> KCI, <NUM> CoCl<NUM>, <NUM>µg/mL of MjTET and <NUM> Leu-pNA as substrate. The given pH values are at <NUM>. Incubation was done at <NUM> for <NUM> and the quantity of produced pNA was evaluated by measuring the absorbance at <NUM>.

The metal cations XCl<NUM> were incubated at <NUM> concentration with <NUM>µg/mL of MjTET and <NUM> of Leu-pNA in <NUM> CHES, <NUM> KCI, pH <NUM> for <NUM> at <NUM>. To assess the effect of metal cations on MjTET activity, the enzymatic reaction was followed as described before.

Different temperatures ranging from <NUM> to <NUM> were tested on MjTET activity. Incubation was done in <NUM> CHES, <NUM> KCI, <NUM> CoCl<NUM>, pH <NUM> in presence of <NUM>µg/mL of protein and <NUM> Leu-pNA for <NUM>. Absorbance of released pNA was measured at <NUM>.

The effect of temperature on MjTET stability was assayed by incubating the enzyme (<NUM>µg/ml) at <NUM>. Aliquot of <NUM>µl was taken at different time intervals, and the remaining aminopeptidase activity was assayed against <NUM> Leu-pNA in <NUM>µl of <NUM> CHES, <NUM> KCI, <NUM> CoCl<NUM>, pH <NUM> incubated at <NUM> for <NUM>. Enzymatic stability of MjTET was evaluated by measuring the quantity of produced pNA at <NUM>.

The inhibitory effect of EDTA, Amastatine and Bestatine was tested on the MjTET enzymatic activity. <NUM>µg/mL of MjTET were incubated with each inhibitor separately in the presence of <NUM> Leu-pNA, <NUM> CHES, <NUM> KCI, <NUM> CoCl<NUM>, pH <NUM> at <NUM> for <NUM>. Three replicates (with inhibitor) and two controls (without inhibitor) were assayed for each experimental point.

The hydrolytic activity of MjTET on synthetic chromogenic and fluorogenic compounds was determined by using different aminoacyl-pNAs and aminoacyl-AMCs through the following procedure. Reactions were initiated by addition of <NUM>µg/mL of enzyme to a pre-warmed mixture containing <NUM> of the chromogenic or fluorogenic compound in <NUM> CHES, <NUM> KCI and <NUM> CoCl<NUM>, pH <NUM>, in a total volume of <NUM>µl. Incubation was performed at <NUM> for <NUM>. The quantity of liberated pNA was evaluated by measuring the absorbance at λ=<NUM> in a JASCO V-<NUM> spectrophotometer, and for AMC by measuring the fluorescence using excitation and emission wavelengths of <NUM> and <NUM>, respectively in a JASCO FP-<NUM> spectrofluorometer. Three replicates and two enzyme blanks were assayed for each experimental point. Additional enzymatic studies were performed in order to investigate MjTET capacities to hydrolyze longer substrates (H-Trp-Phe-Tyr-Ser(PO<NUM>H<NUM>)-Pro-Arg-pNA (SEQ ID NO : <NUM>), H-Met-Trp-Ala-Glu-Asn-Lys (SEQ ID NO:<NUM>), H-Tyr-Asp-Thr-Ser-Ile-Val-Gln-Arg (SEQ ID NO:<NUM>), H-Asp-Ala-Tyr-Pro-Ser (SEQ ID NO: <NUM>) and H-Glu-Gly-Ile. <NUM>µg/ml (final concentration) of MjTET were added to a pre-warmed mixture of <NUM> peptide, <NUM> CHES, <NUM> KCI, <NUM> CoCl<NUM>, pH <NUM>. To avoid water evaporation, <NUM>µl of mineral oil were added on the top of the total volume. The reaction incubation was done at <NUM> during <NUM>. Aliquot of <NUM>µl was then removed and added to <NUM>µl of <NUM> % acetonitrile (ACN), <NUM> % trifluoroacetic acid (TFA). Proteins were removed by centrifugation at <NUM>,<NUM> rpm during <NUM>. <NUM>µl of the supernatant were retrieved and injected on Nova-Pak C18 column, (<NUM>, <NUM> x <NUM>, (Waters)) in a HPLC purifier system (PerkinElmer), equilibrated with <NUM> % ACN, <NUM>% TFA. The elution of peptide products was achieved with a linear ACN gradient (<NUM>-<NUM> %) and followed by measuring the absorbance at <NUM>. Chromatographic runs were carried out at room temperature.

MjTET-Nt was purified as described above and concentrated up to <NUM>/ml using an Amicon Ultra <NUM> kDa cut/off filter. Crystals were grown at <NUM> in Trisodium citrate <NUM> pH <NUM>, Tertbutanol <NUM> % using the hanging drop vapor diffusion method. MjTET-Nt was mixed with an equal volume of the crystallization solution and crystals grew up to average <NUM> size in two days. The crystals were cryo-protected with Trisodium citrate <NUM> pH <NUM>, Tertbutanol <NUM> % supplemented with <NUM>-Methyl-<NUM>,<NUM>-pentanediol <NUM> %. The crystals were flash cooled in liquid nitrogen prior to data collection at <NUM>. Data set was collected at ESRF beamline ID29. The diffraction extended up to <NUM>Å. Data were indexed and integrated by XDS (<NPL>). Integrated intensities were scaled and merged with SCALA and TRUNCATE from the CCP4 programs suite (<NUM>). MjTET-Nt
<IMG>.

structure was determined by molecular replacement using PHASER MR (<NUM>) and PhTET2 as searching model (PDB ID: 1XFO) depleted of side chains. A clear solution was determined with a Translation Function Z-score of <NUM>. The asymmetric unit contains four monomers and the space group is cubic P2<NUM><NUM>. The model was manually completed and improved in COOT prior to refinement with PHENIX (<NUM>-<NUM> version) software package (Adams et al. The model was then optimized through iterative rounds of refinement and model building resulting in final R-work= <NUM> and R-free= <NUM> (Table <NUM>).

Table <NUM>: Data collection and refinement statistics of MjTET-Nt(-_Zn<NUM>) and MjTET(Zn<NUM>_Zn<NUM>). Values in parenthesis correspond for the highest resolution shell. <MAT> where Ihkl, j is the jth intensity measurement of reflection hkl and < I > is the average intensity from multiple observations. <MAT> with Ihkl,j is the jth intensity measurements of reflection hkl and < I > is the average intensity from multiple observations. n represents the multiplicity of the measurements.

CC<NUM>/<NUM>= correlation coefficient between random half datasets. <MAT> for all data except <NUM>% which were used for Rfree calculation.

During the initial step of refinement, the inventors realized that MjTET-Nt structure was depleted of Zn<NUM>+ in Site1. Consequently, in order to provide description of MjTET active site, MjTET-Nt crystals were soaked with Zn<NUM>+ excess solubilized in the cryo-protection solution used above. Data at <NUM>Å resolution were collected at ESRF beamline ID29. Data processing and refinement were conducted as described above, except that the <NUM>Å resolution MjTET-Nt(-_Zn1) model was used as reference model to steer refinement of MjTET(Zn1_Zn2) resulting in final R-work= <NUM> and R-free= <NUM> (Table <NUM>).

Four microliters of sample was applied to <NUM>:<NUM> Quantifoil holey carbon grid (Quantifoil Micro Tools GmbH, Germany) and the grid was frozen in liquid ethane with a Vitrobot Mark II (FEI, the Netherlands). The sample was observed with FEI Polara at <NUM> kV. Images were recorded on K2 summit direct detector (Gatan Inc. , USA) in super resolution counting mode. Movies were recorded at a nominal magnification of <NUM>,<NUM> (<NUM>Å/pixel at the camera level) for a total exposure of <NUM> and <NUM> per frame resulting in <NUM> frame movies with a total dose of ~<NUM> e-/Å<NUM>. <NUM> movies were manually recorded with Digital Micrograph (Gatan Inc.

Movies were first motion corrected with unblur (<NPL>). Around <NUM> particles were picked semi automatically with boxer from the EMAN suite (<NPL>) and 2D classified in <NUM> classes in RELION <NUM> (<NPL>). The bestdefined 2D class averages were used to generate an initial model using the RICO server imposing tetrahedral symmetry. The resulting low-resolution reconstruction was used as a template for automatic particle picking using the Fast Projection Matching (FPM) method (<NPL>) as described in (<NPL>). To speed up calculation, the raw micrographs were binned by a factor of <NUM> (<NUM>Å/pixel) and, during template matching, the resolution was limited to <NUM>. For each micrograph, only particles having cross correlation with the reference higher than the mean correlation of all the particles were kept. The output coordinate files from FPM were converted to boxer one (. box) and imported in RELION <NUM> where all subsequent steps were done with two-times binned images (final sampling of <NUM>). CTF estimation, particle extraction and preprocessing were done in RELION. The data set was first cleaned by 2D classification. A first 3D auto-refine was then done using the ab-initio model obtained with the RICO server imposing tetrahedral symmetry. The obtained 3D model was used as input for 3D classification in five classes. The particles belonging to the classes showing the best resolved features were used to do a final 3D auto-refine. The final reconstruction includes <NUM> out of <NUM> and has a resolution of <NUM>Å at FSC = <NUM>.

To gain insight into the basic aspects of TET structure and function, the inventors cloned and expressed in E. coli MJ0555 gene encoding for the single putative TET peptidase that is present in the genome of M. jannaschii. The corresponding recombinant protein was purified with protocols inspired from those developed for the <NUM> hyperthermophilic TET from P. horikoshii. When analyzed by gel filtration chromatography, MJ0555 protein displayed an apparent molecular mass similar to one determined for the homologous TET dodecameric proteins from P. horikoshii and P. Negative stain electron microscopy images of MJ0555 samples showed typical hollow-tetrahedral dodecameric particles with dimensions similar to those previously determined for the different M42 TET peptidases and MJ0555 was therefore named MjTET (<FIG>).

The sequence alignment of MjTET with other TET peptidases from hyperthermophilic archaea revealed ~<NUM>% sequence identity with PhTET2 that was defined as leucine metallo-aminopeptidase (LAP). The optimal conditions for MjTET activity were therefore tested using Leu-pNA. To assess for the metallic dependent behavior of MjTET, the inventors tested several inhibitors of M42 metallopeptidases. Under optimal conditions of MjTET activity (see bellow), adding <NUM> EDTA to the reaction volume completely inhibited the enzyme, thus confirming the metallic dependence of MjTET. Moreover the MjTET LAP activity was completely inhibited by <NUM> amastatine and <NUM> bestatine, respectively aminopeptidase and leucyl aminopeptidase inhibitors.

The inventors investigated the effect of pH on MjTET LAP activity. <FIG> shows that MjTET maintained high percentage of activity (between <NUM> to <NUM>%) over a wide range of pH, from neutral (pH <NUM>) to alkaline (pH <NUM>). Moreover it maintains more than <NUM>% of its activity in acidic and alkaline conditions such as pH <NUM> and <NUM>. Thus as PhTET2, MjTET is an aminopeptidase that can work on broad range of pH conditions (between <NUM> and <NUM>, for PhTET2). As different divalent metal ions have been found to modulate the activity of aminopeptidases, the inventors tested the effect of several metals on MjTET activity. The data presented on <FIG> showed that the increase of LAP activity occurs only in the presence cobalt ions. By contrast, a significant inhibition was observed when <NUM> ZnCl<NUM> was added to the reaction mixture. Thus, MjTET can be defined as cobalt-activated enzyme.

The temperature dependence of MjTET activity was determined. As shown in <FIG>, LAP activity was found to be maximal at <NUM>, corresponding to the optimal growth temperature of M. jannaschii species. When incubated at this temperature, MjTET showed a half-life of eight hours. No activity was detected in mesophilic conditions under <NUM>.

A common hallmark to M42 TET aminopeptidases studied so far is their marked preference for a subset of amino acids. Hence, in P. horikoshii, PhTET1 exhibits narrow selectivity toward glutamyl and aspartyl residues while PhTET2 is a leucyl aminopeptidase with broad activity against neutral amino acids and PhTET3 is a lysyl aminopeptidase with clear preference for positively charged residues (<NUM>-<NUM>). In order to determine MjTET function, its amino acid selectivity was investigated using chromogenic and fluorogenic monoacyl substrates. As shown in <FIG>, the enzyme acted on large number of aminopeptidase substrates. The optimal amidolytic activity was observed against Leu-pNA, thus allowing classifying MjTET as leucine aminopeptidase. Interestingly, MjTET also displayed significant amidolytic activities, principally against hydrophobic residues such as phenylalanine, methionine and tyrosine. Reduced activities were also detected in the presence of polar and positively charged residues. Aspartate and glutamate, cysteine and proline were the only residues toward which no activity was detected. Finally, no activity was detected against blocked residues and residues in D-stereo configuration, as shown for Ac-Leu-pNA and D-Leu-pNA substrates, respectively. M42 aminopeptidases displayed poor amidolytic activity against aromatic substrates such as phenylalanine and tyrosine. PhTET2 was found to exhibit weak activity against aromatic amino acids, however, as shown in <FIG>, MjTET enzymatic activities appeared to be more efficient notably against phenylalanine. In addition to the broad activity spectrum, this represents another noticeable difference between MjTET and PhTET2.

The exopeptidase activities and specificities can be different in peptide context, compared to the one measured on monoacyl compounds. To test if MjTET maintains broad amino acid substrate specificity as well as the ability to cleave aromatic residues in peptide context, its action was studied on different substrates containing <NUM> to <NUM> residues. After incubation with MjTET enzyme, the peptide composition of the reaction mixture was investigated at different time points by reverse phase HPLC. <FIG> shows the hydrolysis profile of the H-Trp-Phe-Tyr-Ser(PO<NUM>H<NUM>)-Pro-Arg-pNA peptide. After <NUM> minutes, the original peptide substrate was almost hydrolyzed and four peptide products were detected, thus confirming that the enzyme displays broad aminopeptidase activity, even on peptides containing aromatic residues. This was confirmed with other substrates: H-Asp-Ala-Tyr-Pro-Ser (<FIG>), H-Tyr-Asp-Thr-Ser-Ile-Val-Gln-Arg, H-Met-Trp-Ala-Glu-Asn-Lys, and H-Glu-Gly-Ile (data not shown). Interestingly, these peptides also contained Asp and Glu residues. Since no activity was measured against Asp-pNA and Glu-pNA, it can be inferred that, these negatively charged amino acids can also be cleaved by MjTET in peptide context, indicating that C-terminal end is required for better fitting of the P1 amino acid inside "S1" specificity pocket of the enzyme. Taken together these experiments showed that, compared to other M42 aminopeptidases, MjTET is characterized by unusual broad substrate specificity.

Crystal structure of MjTET-Nt was resolved at <NUM>Å by molecular replacement using PhTET2 monomer structure (PDB ID: 1XFO) (<NUM>) as search probe. The asymmetric unit of the crystal encloses four monomers, each of ~<NUM> kDa and made up of <NUM> amino acids. The monomer had typical structure of M42 aminopeptidases forming TET dodecamers. Comparison of the dimerization and oligomerization interfaces of MjTET and PhTET2, by using PISA server (http://www. uk/pdbe/pisa/), revealed that they are very similar and involve the same monomer contacts with nearly the same number of hydrogen bonds and salt bridges.

MjTET monomer can be divided in two α/β globular domains connected by two loops (residues <NUM>-<NUM> and <NUM>-<NUM>) (<FIG>). The bigger domain is the catalytic one formed by residues <NUM>-<NUM> and <NUM>-<NUM> and contains the N- and C-terminal ends. The smaller one is the dimerization domain (also named dimerization domain I) formed by residues <NUM>-<NUM>. No electron density was observed for His-tag Nt neither for <NUM>-<NUM> residues. The catalytic domain contains central eight-stranded β-sheet surrounded by seven α-helices and two <NUM><NUM> helices. The two β-strands located on each side of this β-sheet are antiparallel and shorter than the central four β-strands, which are much longer and parallel. Additionally, three-stranded antiparallel β-sheet (residues <NUM>-<NUM> and <NUM>-<NUM>) is positioned on the surface of the catalytic domain and can be named dimerization domain II. The dimerization domain I slightly exhibits cylindrical β-barrel shape made up of six antiparallel β-strands and two α-helices and one <NUM><NUM> helix.

The cleft located between the catalytic and dimerization (I) domains harbors the binuclear active site of the metallo-aminopeptidase. In untreated MjTET-Nt crystals, only one metal ion per monomer of protein was detectable (MjTET-Nt(-_Zn2)). The second exchangeable metal (M1) was detected after soaking MjTET-Nt crystals with excess of zinc ions during cryoprotection step. In this condition, two Zn<NUM>+ ions are modeled in the active site of the enzyme (MjTET-Nt(Zn1_Zn2)). Zn<NUM>+<NUM> has a trigonal bipyramidal geometry and is coordinated by Asp175Oδ2, Glu208Oε1,ε2, His321Nε2 and Citrate Oβ2. However, Zn<NUM>+<NUM> is tetrahedrally coordinated by His62 Nε2, Asp175Oδ1 and Asp230Oδ1,δ2 (<FIG>). The distance between the metal ions is ~<NUM>Å comparable to that one of all other M42 TET peptidases and the leucyl aminopeptidase AAP of Vibrio proteolyticus (formerly known as Aeromonas proteolytica) used as prototype to describe the enzymatic mechanism of peptide bond hydrolysis by binuclear aminopeptidase (<NUM>).

Behind the active site is located the specificity pocket "S1" that was suggested to receive the side chain of the residue that will be cleaved (<NUM>). A mainly large and hydrophobic specificity pocket (residues <NUM>-<NUM>, <NUM>, <NUM>, <NUM>-<NUM>, <NUM>, <NUM>, <NUM>, <NUM>-<NUM>, <NUM>) was found in MjTET monomer, which is in agreement with the preference of this aminopeptidase to hydrolyze hydrophobic residues such as leucine and methionine as described above. Interestingly, this pocket is characterized by the presence of glycine residues (Gly256, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>) that may provide important flexibility to this region, thus allowing the reception of large side chains such as those of aromatic residues especially phenylalanine and tyrosine. Furthermore, negatively charged amino acids, such as Asp259 and Glu289, also present in the "S1" pocket could interact with the positive side chains of histidine, lysine and arginine for potential cleavage by MjTET (<FIG>).

Previous structural studies showed that, despite their similar architectures, the different types of TET complexes showed differences in their secondary and tertiary structures as well as pores and channels dimensions (<NUM>). Noticeable changes were also detected in the surface charge properties of the different TET edifices, in particular with respect to the electrostatic charge distribution in the specificity pocket. These differences were proposed to be responsible for the contrasted substrate specificities between the different TET enzymes and allowed to propose models for peptide trafficking inside the particle (<NUM>,<NUM>,<NUM>,<NUM>). However, there is a lack of structural information on the TET particle interior, apparently due to flexible regions that could not be completely resolved by X-ray crystallography, which preclude validating the proposed models. In this work, the inventors combined X-ray crystallography and cryo-EM experiments to provide better insights into the general internal organization of TET particles.

Intriguingly, electron density was usually absent for the loops harbored by the dimerization domain (I) in crystal structures of TET complexes, especially those belonging to M42 family. This observation indicated the mobile behavior of these loops and may suggest an important role in substrate trafficking. In MjTET cryo-EM structure, electron densities of the twelve loops (residues <NUM>-<NUM>) are clearly visible allowing us to build the X-ray structure missing parts (<FIG>). In fact, to get the atomic structure of X-ray missed loops, the inventors fitted the X-ray structure of MjTET dimer (building block) in cryo-EM map resolved at <NUM>Å, wherein the inventors built up the internal loop residues (<NUM>-<NUM>) of the dimers, by using Coot software (<NUM>). Then, the inventors generated automatically the complete tetrahedral particle for which the inventors checked the position of all constructed internal loops. The analysis of complete particle structure achieved by combining crystallography and cryo-EM, revealed new features regarding internal organization of tetrahedron. Indeed, the inventors showed here that MjTET entire loops established internal bulk inside the dodecameric macromolecule (<FIG>). MjTET electrostatic potential showed strong positive charge engendered by these internal loops. They appeared to disrupt peptide trafficking inside the particle. Interestingly, these positive patches are located behind negatively charged one created by acidic residues of three building blocks forming the opening pore of the particle (<FIG>). These negative charges were described previously to captivate positively charged N-termini of peptide substrate. The location of internal positively charged loops behind the negative patches might avoid peptide navigation from entry pore to the exit hole located on opposite side of the tetrahedron. Furthermore, the organization of the dynamic loops in the same building block, showed that each loop extended near the active site of adjacent monomer, with a peculiar orientation of His116 toward this neighboring site (<FIG>). Together, the newly defined positive network that obstructed the internal cavity of MjTET particle and the loop orientation toward active site provide a significantly shorter route from the outside of TET to one of the three catalytic chambers present near the entry hole compared to the alternative route that crosses the central cavity to catalytic chamber situated at the opposite side of the tetrahedron (<FIG>).

Genomic survey and functional characterization indicate that in prokaryotic cells the number of TET proteins can vary from one to up to four different versions. In the deep-sea hyperthermophilic archaeon P. horikoshii, three different versions of TET complexes having contrasted substrate preferences have been described (<NUM>,<NUM>,<NUM>,<NUM>). According to MEROPS classification, M. jannaschii, another hyperthermophilic archaeon isolated from a natural environment similar to the one of P. horikoshii, contains only one gene encoding for a putative M42 family TET enzyme of MH clan. In this work, the inventors showed that the corresponding recombinant protein, called here MjTET, is indeed a TET aminopeptidase. The search for its physiological function using monoacyl and peptide substrates revealed the enzyme is a leucine aminopeptidase able to cleave hydrophobic, neutral as well as aromatic and positively charged residues such as phenylalanine, tyrosine, lysine and histidine. MjTET can therefore be qualified as non-specialized peptidasome. horikoshii, M. jannaschii is an autotrophic organism that does not rely on the hydrolysis of external peptide to support its metabolism. Thus, the inventors can speculate that the presence of single TET system, "a priori" less efficient than the multiple specialized TET enzymes found in Thermococcales, could be related to autotrophy.

PhTET2 was also described as leucine aminopeptidase. It shares ~<NUM>% of sequence identity with MjTET. However, its specificity range is not as wide as the one of MjTET and it is devoid of hydrolytic activity toward N-terminal phenylalanine residues. Therefore, the comparison of the two enzyme 3D structures can help to determine the structural determinants responsible for the remarkable MjTET polyvalence. This analysis showed that the "S1" specificity pocket of MjTET enzyme is slightly larger than PhTET2 one, and is enriched by glycine residues that are not present in other specialized TET enzymes. Compared to PhTET2, MjTET "S1" pocket contains three additional residues and three glycine residues that could confer more flexibility thus allowing accommodating phenylalanine and tyrosine large side chains. Compared to MjTET, the reduce activity of PhTET2 against positively charged substrates could be explained by the presence of Lys261 (in reference to PhTET2 sequence) that can disrupt positive side chain fitting in "S1" pocket. In peptide context, MjTET also displayed significant cleavage capacity of acidic residues. Colombo et al. revealed the presence of third metal ion in the specificity pocket of PfTET3, a lysyl aminopeptidase of P. furiosus (<NUM>). This cation is coordinated by Thr232 and Glu281 residues and facilitate the accommodation of negative charged side chain of substrates in "S1" pocket and subsequently their degradation (<NUM>). In similar fashion, the "S1" pocket of MjTET encloses Thr232 and Glu289, which are localized in the same position as the former ones, allowing the possibility to receive a third metal, thus extending MjTET cleavage capacity to acidic residues.

The cryo-EM experiments allowed the edification of the twelve internal dynamic loops carried by the dimerization domains inside the M42 MjTET particle cavity. The loop positions with respect to the active sites, entry holes and their topology within the central cavity suggest that they are of pivotal importance for the functioning of the TET dodecamers. In particular, the cryo-EM map revealed that some internal loops residues, particularly His116, are orientated toward the binuclear active site. Sequence alignments reveal a strict conservation of His116 amongst M18 and M42 aminopeptidases exhibiting tetrahedral architecture (<FIG>). In eukaryotic M18, His170 of hDNPEP (human aspartyl aminopeptidase) and His166 of bDNPEP (bovine aspartyl aminopeptidase) are localized near the active site and were proposed to be involved in substrate binding and in the stabilisation of tetrahedral intermediate of enzymatique reaction (<NUM>,<NUM>). Moreover, the His170Phe mutation completely abolished the enzymatic activity of hDNPEP (<NUM>). Thus, in M42 TET, histidine residue located at the internal loop extremity is suggested to play important role in peptides hydrolysis. Interestingly in cryo-EM structure, the distance that separates the His116 from the active site is much longer than the one measured in the crystal structures of M18 family TET (characterized by longer amino acid sequence than MjTET one). This may reflect a dynamic movement of MjTET internal loops to be closed to the active site. Thus, this motion allows the orientation of N-ter polypeptide end toward the binuclear catalytic site where hydrolysis will occur.

The position of the internal loops as determined the MjTET particle leads to drastically change our vision regarding the peptide navigation pathway within M42 family TET particles. The twelve internal loops of MjTET built in the electron density of cryo-EM map obstructed the internal cavity. They define a strong positively charged cluster generated by Lys113, His116, Arg117, Lys119, Lys123, Lys125 and Lys128 residues. These features would prevent polypeptide substrates to cross the particle from one entry hole to the opposite apex where three active sites are present, as the inventors proposed before for the three TET from P. horikoshii. Thus, another route for peptide navigation must be considered. The analysis of MjTET surface electrostatic potential showed three well-defined negative patches located at the entry hole of the tetrahedron. The comparison of the electrostatic potential of the different thermophilic M42 family TET complexes revealed that this feature is well conserved. In MjTET, each acidic patch is made up by six acidic amino acids, Asp74-<NUM>, Glu243, Asp244 and Asp248 of each dimer loop encompassing the large opening of the particle. The inventors noticed that these loops situated at the entrance hole and which harbored precedent negatively charged residues are located in front the internal positive loops at a correct distance and position allowing their interaction. Therefore, the function of the entry loops may be polypeptide capture in such a manner that the substrate N-terminal will be pre-orientated toward the internal cavity and one of the twelve internal positively charged loop devoted to substrate guidance and positioning in the catalytic chamber.

In conclusion, our results indicated that TET combines four peptide-processing modules each consisting in one entry hole in connection with three adjacent catalytic chambers. Three structural loops form a chain to guide the substrate N-termini toward one of three active sites of three adjacent catalytic chambers. The building of these modules also involves three other loops that participate to another functional module, which underline the functional relevance of TET dodecamerization. In this new model, and unlike to the previously proposed one, the four tetrahedron apexes could process simultaneously, and in an independent manner, at least four peptides while avoiding peptide congestion inside the particle.

In order to evaluate the effect of the temperature on the activity of MjTET, the inventors evaluated the relative amount of intact peptides further to an incubation with the TET protein at different temperatures.

The inventors have measured the relative amount of intact peptide further to an incubation with MjTET.

MjTET activity was measured on the following peptide : YFLMNENRYTSWNSE (SEQ ID NO: <NUM>). The inventors evaluated the time necessary to hydrolyse <NUM>% of the peptide at different tempertaures.

Results are shown in the following table :
<IMG>.

Claim 1:
Use of a TET protein as a N-terminus aromatic amino acid residues specific exopeptidase, said TET protein consisting of the amino acid sequence as set forth in SEQ ID NO: <NUM>,
or any homologous protein derived from said TET protein as set forth in SEQ ID NO: <NUM> by substitution, addition or deletion of at least one amino acid, provided that the derived protein retains at least <NUM> % of identity with the amino acid sequence as set forth in SEQ ID NO: <NUM>, and said derived protein retaining a N-terminus aromatic amino acid residues specific exopeptidase activity.