SpsB was also restored to its active form by mutating residue 36 back to the native Ser, and the C-terminal residues —, which were disordered in the uncomplexed structure, were replaced with a Strep -tag II to aid purification. Using this tethering strategy, SpsB was crystallized with three different peptides Table 2. Two Pep1 and Pep2 are substrate peptides with very different C-region sequences which are actively cleaved by our SpsB constructs Ting et al. Table 2 Peptide sequences. All three SpsB—peptide complex structures were determined at resolutions of 1. Residues P2—P5 of all three peptides bind in an identical fashion, and all SpsB residues that contact Pep1 and Pep2 are also identically positioned [r.
The Ala side chains of peptide residues P1 and P3 occupy hydrophobic depressions that constitute the S1 and S3 specificity pockets, respectively Fig. In the Pep1 and Pep2 substrate complexes, the P1 methyl group makes intimate contacts with the side chains of Ile32, Met37 and Val76 in the S1 pocket, with main-chain atoms from Lys33, Gly34, Ser36 and Met37 also at a van der Waals distance. In the Pep3 structure, the P1 methyl group does not protrude as far into the S1 pocket, with an average distance of 4. The S3 pocket is broader, with an average distance between the P3 methyl group and the residues that line the S3 pocket for all three peptide structures of 4.
This is consistent with observed signal-peptide sequence variability at P3, which allows the pocket to accommodate larger aliphatic residues. The side chains of the P2 and P4 peptide residues remain solvent-exposed and make no contact with the enzyme, in accordance with predictions from the LepB structure Paetzel et al. Only one other hydrogen bond is apparent, linking the P5 proline carbonyl O atom to the peptide N atom of Thr The positioning of the proline orientates the peptide chain so that no further residues make main-chain contacts with the enzyme Fig. The Pep1 structure shows that the peptide is similarly directed away from the enzyme at P5 when the residue is histidine Fig.
The SpsB construct used here had previously been shown to be fully active, cleaving signal peptides to release a mature protein Ting et al. This implies that the lack of density for the mature protein portion of the cleavable peptides Pep1 and Pep2 is owing to enzymatic cleavage and not to disorder of the peptide in the crystal structure.
The Ser36 hydroxyl points away from the cleaved peptide, which is the result of a clash with the C-terminus of the cleaved peptide Fig. The P1 carbonyl O atom makes two hydrogen bonds Fig. Protein—peptide interactions in the active site support the catalytic mechanism proposed for SPases Paetzel et al. Any additional residues in the mature portion of the pre-protein should project away from the enzyme. Signal peptides are critical N-terminal extensions that function as an address code for proteins, with cleavage of the signal peptide by a signal peptidase the last step in protein secretion.
Type I signal peptidases all recognize a central Ala- X -Ala motif that precedes the cleavage site, but the sequence diversity outside this canonical motif is such that ambiguity remains as to how the enzyme can process hundreds of different peptides while retaining strict fidelity. A complicating factor is that enzymes from different bacteria have varied susceptibility to SPase I inhibitors. While there is a large variation in the size and structure of the noncatalytic domain, the core catalytic domains are very similar. Importantly, the peptide-binding clefts of the two enzymes are virtually identical and, as predicted by the amino-acid sequence, have matching catalytic apparatus.
In the present study, the SpsB—inhibitor peptide Pep3 complex represents a model for the pre-protein—enzyme Michaelis complex, whereas the substrate peptides Pep1 and Pep2 give a picture of the post-cleavage state. There is little change in the peptide-binding cleft between the uncomplexed and peptide-bound SpsB structures. In the uncomplexed structure Tyr30 partially occupies the S3 pocket but moves when peptides bind, and Val76, which forms a bridge between the S1 and S3 pockets, adopts an alternative rotamer. These changes are consistent with changes observed between the apo and inhibitor-bound structures of E.
Protein—peptide interactions in SpsB support the proposed catalytic mechanism for SPases, with nucleophilic attack by the serine Ser36 on the si face of the scissile peptide bond Paetzel et al.
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Like LepB, the SpsB peptide-binding cleft contains two shallow pockets, designated the S1 and S3 substrate-binding pockets, which accommodate the methyl side chains of the P1 and P3 Ala residues. The high level of conservation in and around the two pockets is also consistent with peptide-binding studies showing that alteration of the residues that bridge between the two pockets in LepB leads to slippage in the site of peptide cleavage and alteration of the S3 pocket specificity Karla et al.
In our structures the side chains of the P2 and P4 peptide residues remain solvent-exposed. This contradicts predictions from in silico modelling of LepB Choo et al. Significantly, it is the core Ala- X -Ala motif that provides the most substantial peptide—enzyme interaction Fig. In the Pep2 and Pep3 structures, the P5 proline directs the peptide chain away from the enzyme, with no residues prior to P5 making contact with the enzyme. A proline is commonly found at P5 in S. A number of factors in the enzyme—peptide substrate complex suggest that it is optimized to enhance product turnover.
These include the minimal degree of interaction between the peptide and enzyme and the conserved binding mode of different peptide substrates. The shallow nature of the substrate-binding cleft, in which the main-chain atoms of the peptide substrate remain solvent-exposed, should also promote rapid association of substrates and dissociation of products, as required for multiple rapid turnovers Tyndall et al.
Looking beyond the P1 and P3 residues, which dominate the peptide-binding interactions and dictate the position of the peptide in the peptide-binding cleft, the P2 and P4 side chains point directly out into the solvent, while the P5 and P6 side chains are accommodated by shallow grooves. These grooves or exposed faces allow the enzyme to bind peptides with highly diverse side-chain composition without affecting fidelity, which is dependent on the Ala- X -Ala motif. The structures presented here, together with signal-peptide sequence and mutagenesis data Jain et al.
While it is not clear whether the cleavage of signal peptides occurs during or after protein translocation, the anchoring of the H region of signal peptides in the membrane appears to be essential for orientating the signal peptide. Only when we were able to mimic this restraint by tethering the N-terminus of our peptide could we successfully co-crystallize SpsB with bound peptides. Gram-positive signal peptides are longer than both Gram-negative bacterial and eukaryotic signal peptides, with the site of cleavage predicted to be on the membrane surface Dalbey et al.
Whether cleavage occurs at the cell surface, as modelled here Fig. However, from our structures we can infer a minimalistic model of peptide recognition in which the core Ala- X -Ala motif both defines specificity and accounts for the majority of the interactions between the peptide and enzyme. Few other residues make specific contacts, and these involve main-chain atoms, independent of sequence, with the divergent side chains accommodated via exposed faces. Barkocy-Gallagher, G. Jr Biochemistry , 39 , — Acta Cryst. D 66 , 12— BMC Bioinformatics , 9 , Suppl.
D 66 , — D 62 , 72— D 67 , — Science , , — Synchrotron Rad. FEBS Lett. This section lists the commands available to monitor the "Flow Recovery Support for Tethering Detection" feature. This section lists all the bulk statistics that have been added, modified, or deprecated to support this feature. This feature can be used to detect tethering for users using stealth tethering applications such as FoxFi, EasyTether, and so on.
The DNS-based solution is implemented to address the deficiencies that other incumbent solutions had in detecting IPv6 tethered flows. Once the flow is classified as tethered from first SYN or mid-flow SYN , that flow will remain tethered throughout the lifetime of the flow. This feature provides the capability to limit the number of SYN packets that need to be analyzed on the same flow. The max-syn-packet-in-flow command in the ACS Rulebase Configuration mode is configured to limit the maximum number of SYN packets to be analyzed for tethering detection.
The flow end-condition tethering-signature-change command in the ACS Rulebase Configuration mode is configured to control the behavior of mid-flow EDR generation due to tethering signature change. The Tethering Detection feature is enhanced to support tethering detection bypass based on Interface ID. Only one Interface ID can be configured for tethering detection bypass in the active-charging service. IPv4 tethering detection and UA-based tethering detection is not impacted. The Tethering Detection database files must be populated and loaded on to the ASR chassis by the administrator. The procedure to load the database is the same for all the different databases.
Before the database s can be loaded for the first time, tethering detection must be enabled using the tethering-database CLI command in the ACS Configuration Mode. For all the databases, only a full upgrade of a database file is supported. Incremental upgrade is not supported. If, for any particular database, the upgrade procedure fails, the system will revert back to the previous working version of that database. This can be used to collect os-signatures that can then be used to build an OS database for the tethering detection feature.
This section provides an overview of loading and upgrading the databases used in tethering detection. Any further upgrades to the database files can be done by placing the file named new-filename in the designated directory path. ACS auto-detects the presence of files available for upgrade daily. When a new version of a file is found, the upgrade process is triggered. The upgrade can also be forced by running the upgrade command in the CLI. On a successful upgrade this file is renamed to filename. This section describes the Tethering Detection configuration.
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The following examples illustrate two different implementations of the Tethering Detection configuration. The following type of configuration is suitable where ECS performance is critical and the operator wants to put in a flat charging plan in place for all the tethered traffic.
In such a scenario, addition of a single new ruledef to the configuration suffices. The introduction of an additional coordination group in the bidentate spiro aminophosphine ligand L6 led to a very stable and efficient catalyst for the AH of simple ketones 27, affording the chiral alcohols 28 in up to Moreover, chemo-, regio- and stereoselectivity can often be different from that of AH.
In the ATH process, the transition-metal catalyst is able to abstract a hydride and a proton from the hydrogen donor and deliver them to the carbonyl moiety of the ketone. A major drawback of using i -PrOH is the reaction reversibility, giving limited conversions and affecting the enantiomeric purity of the products after long reaction times. The use of formic acid can overcome these drawbacks, although only a narrow range of catalysts that tolerate formic acid is available.
In parallel with the discovery of efficient ruthenium catalysts for AH, Noyori and co-workers found a prototype of chiral arene Ru II catalysts of type C8 bearing N -sulfonated 1,2-diamines e. It was first disclosed by Noyori, that a N-H moiety is necessary for an efficient transfer of hydrogen from the metal hydride. In contrast, H 2 -hydrogenation is less successful when using this system.
There is a continuing search for stable catalysts that would not degrade easily during the hydrogenation process, thus making it possible to execute as many as possible catalytic cycles. The complexes C11 and C12 were not isolated but used in situ. In this respect the development of catalysts with similar properties to replace platinum-group metals is very desirable from both the economic and environmental points of view.
In fact, iron is cheap and ubiquitous, and its traces in final products are not as serious a problem as traces of ruthenium, for example Morris, A range of aryl, alkyl, cyclic, heterocyclic, and aliphatic ketones were hydrogenated under 50 bar of H 2 with a combination of inexpensive Cu OAc 2 and monodentate binaphthophosphepine ligand L17 Junge et al.
Owing to the stronger bonding of Os compared to Ru, robust and thermally stable complexes can be obtained, which is important for achieving highly productive catalysts. The main disadvantage, however, is the low solubility of the homogenous metal catalysts and most of the organic substrates when going from organic to aqueous media, which may be reflected in a reduced activity and selectivity. To circumvent this, either hydrophilic, often charged, functionalities can be introduced to ligands to render the catalysts water-soluble, or different surfactants can be added in order to solvate the reaction partners, although in some cases water-insoluble catalysts can deliver a superior activity and selectivity.
The latter catalyst system appeared to be quite stable, since it could be recycled six times with little loss of performance. Similarly, an in-situ -prepared catalytic complex from the proline-functionalized ligand L21 and [RuCl 2 p -cymene ] 2 in a ratio showed good activity for the aqueous ATH of acetophenone-type ketones as well as bicyclic ketones Manville et al.
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The tethered Rh complex C20 reported by Wills acts as a very productive catalyst for aqueous-reduction as it continues to turnover a reaction at low loadings, even at 0. The chiral aqua Ir III -complex C21 bearing non-sulfonated diamine was shown to be very flexible in the ATH of -cyano- and -nitroacetophenones as the reaction can be conducted at pH 2 formic acid as well as at pH 5. Surfactants are often added as co-solvents to obtain a sufficient solubility of the reactants, products and metal catalysts, thus retaining the activity and selectivity of the hydrogenation process.
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It is notable that catalysts embedded in these micro-reactors can be separated from the organic phase and reused for at least six times without any loss of activity and enantioselectivity. In recent years ionic liquids ILs have attracted an increasing interest because of their non-volatility, non-flammability and low toxicity. Additionally, ILs are capable of immobilizing homogenous catalysts and facilitating the recycling of catalysts. Ideally, organic products can be easily separated by extraction with a less polar solvent and the IL phase containing catalyst can be reused.
Such an immobilization of catalysts also promises to prevent the leaching of toxic metals into the organic products, which is especially desirable in the production of pharmaceutical intermediates. In contrast, the catalyst activity showed a remarkable drop with each cycle, and therefore the reaction times had to be prolonged for high conversions. Homogenous hydrogenation and transfer hydrogenation may be mechanistically closely related because both reactions involve a metal hydride species under catalytic conditions, thus sharing a multistep pathway of hydride transfer to the ketone, i.
Applied only to the transfer hydrogenation, direct hydrogen transfer Meerwein-Ponndorf-Verly reaction from the metal alkoxyide to the ketone without the involvement of metal hydrides proceeding through a six-membered transition state has also been proposed, and is typical for non-transition metals e.
Noyori and co-workers proposed metal-ligand bifunctional catalysis for their Ru catalysts containing chiral phosphine-amine ligands and for arene Ru-diamine catalysts, which consequently resulted in a widely accepted mechanism to be responsible for the highly enantio-selective hydrogenation and transfer hydrogenation of prochiral ketones Noyori et al. The actual catalysts, Ru-hydrides 31 or 34, are usually created in a basic alcoholic solution under H 2 or not at the beginning of the catalytic reaction from the Ru precursors 30 or Note that only the trans -RuH 2 31 is a very active catalyst.
This concerted process results in the formation of an alcohol product and Ru-amido species 32 or The hydride intermediate 31 or 34 is then regenerated either by the addition of molecular hydrogen or by the reverse hydrogen transfer from a dihydrogen source e. The latter step is considered to be a rate-limiting step. The overall process is occurring outside the coordination sphere of the metal without the interacting of the ketone or alcohol with the metal center.
This is known as an outer-sphere mechanism. It is depicted in Fig. Depending on transition-metal catalysts, an ionic mechanism has also been proposed where the proton and hydride transfer occur in separate steps Bullock, The active species in catalytic cycles, Ru-hydride 31 or 34 and Ru-amido complexes 32 or 35 , have not only been detected but also isolated in some cases Abdur-Rashid et al.
The absolute configuration of the alcohol product in AH is determined in the six-membered transition state resulting from the reaction of a chiral diphosphine-diamine-RuH 2 complex with a prochiral ketone Noyori et al. Because the enantiofaces of the ketone are differentiated on the molecular surface of the saturated RuH 2 complex, a suitable combination of the catalyst and substrate is necessary for high efficiency. The prochiral ketone e. In contrast to the outer-sphere mechanism, here the ketone and alcohol interact with the metal center. One is chirally modified supported metals, and the other is the immobilized homogeneous catalyst on a variety of organic and inorganic polymeric materials.
There are also two major reasons for preparing and studying heterogeneous catalysts: firstly, and most importantly, the better and advanced separation and handling properties, and, secondly, the potential to create catalytic positions with an improved catalytic performance. The ultimate heterogeneous catalyst can easily be renewed, reused without of loss of activity and selectivity, which are at least as good or even better than those of the homogeneous analogue.
The immobilization of a homogeneous metal coordination complex is a useful strategy in the preparation of new hydrogenation catalysts. Much effort has been devoted to the preparation of such heterogenized complexes over the past decade due to their ease of separation from the reaction mixture and the desired minimal product contamination caused by metal leaching, as well as to their efficient recyclability without any significant loss of activity. Preferably, Rh, Ir, and Ru complexes have been employed in the hydrogenations of carbonyl functionality Corma et al.
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Chemically different supports have been used for the immobilization of various homogeneous complexes, including polymeric organic and inorganic supports Saluzzo et al. Supports of an inorganic nature are more suitable owing to their physical properties, chemical inertness and stability with respect to swelling and deformation in organic solvents. The above-mentioned properties of the inorganic supports will facilitate the applications of the materials in reactions carried at higher temperatures and their use in continuous-flow reactions.
Immobilization via covalent bonds is undoubtedly the most convenient, but on the other hand, it is the most challenging method for immobilization to perform on such supports Jones et al.