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See detailThe Lys-Asp-Tyr Triad within the Mite Allergen Der p 1 Propeptide Is a Critical Structural Element for the pH-Dependent Initiation of the Protease Maturation.
Chevigne, Andy; Campizi, Vincenzo; Szpakowska, Martyna et al

in International Journal of Molecular Sciences (2017), 18(5),

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of ... [more ▼]

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element. [less ▲]

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See detailInfrared imaging of high density protein arrays.
De Meutter, Joelle; Vandenameele, Julie ULg; Matagne, André ULg et al

in Analyst (2017)

We propose in this paper that protein microarrays could be analysed by infrared imaging in place of enzymatic or fluorescence labelling. This label-free method reports simultaneously a large series of ... [more ▼]

We propose in this paper that protein microarrays could be analysed by infrared imaging in place of enzymatic or fluorescence labelling. This label-free method reports simultaneously a large series of data on the spotted sample (protein secondary structure, phosphorylation, glycosylation, presence of impurities, etc.). In the present work, 100 mum protein spots each containing about 100 pg protein were deposited to form high density regular arrays. Using arrays of infrared detectors, high resolution images could be obtained where each pixel of the image is in fact a full infrared spectrum. With microarrays, hundreds of experimental conditions can be tested easily and quickly, with no further labelling or chemistry of any kind. We describe how the noise present in the infrared spectra can be split into image noise and detector noise. We also detail how both types of noise can be most conveniently dealt with to generate very high quality spectra of less than 100 pg protein. Finally, the results suggest that the protein secondary structure is preserved during microarray building. [less ▲]

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See detailThe proteolytic system of pineapple stems revisited: Purification and characterization of multiple catalytically active forms.
Matagne, André ULg; Bolle, Laetitia; El Mahyaoui, Rachida et al

in Phytochemistry (2017), (138), 29-51

Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies ... [more ▼]

Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies have been reported aiming at the fractionation and characterization of the many molecular species present in the extract, but more efforts are still required to obtain sufficient quantities of the various purified protease forms for detailed physicochemical, enzymatic and structural characterization. In this work, we describe an efficient strategy towards the purification of at least eight enzymatic forms. Thus, following rapid fractionation on a SP-Sepharose FF column, two sub-populations with proteolytic activity were obtained: the unbound (termed acidic) and bound (termed basic) bromelain fractions. Following reversible modification with monomethoxypolyethylene glycol (mPEG), both fractions were further separated on Q-Sepharose FF and SP-Sepharose FF, respectively. This procedure yielded highly purified molecular species, all titrating ca. 1 mol of thiol group per mole of enzyme, with distinct biochemical properties. N-terminal sequencing allowed identifying at least eight forms with proteolytic activity. The basic fraction contained previously identified species, i.e. basic bromelain forms 1 and 2, ananain forms 1 and 2, and comosain (MEROPS identifier: C01.027). Furthermore, a new proteolytic species, showing similarities with basic bomelain forms 1 and 2, was discovered and termed bromelain form 3. The two remaining species were found in the acidic bromelain fraction and were arbitrarily named acidic bromelain forms 1 and 2. Both, acidic bromelain forms 1, 2 and basic bromelain forms 1, 2 and 3 are glycosylated, while ananain forms 1 and 2, and comosain are not. The eight protease forms display different amidase activities against the various substrates tested, namely small synthetic chromogenic compounds (DL-BAPNA and Boc-Ala-Ala-Gly-pNA), fluorogenic compounds (like Boc-Gln-Ala-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC), and proteins (azocasein and azoalbumin), suggesting a specific organization of their catalytic residues. All forms are completely inhibited by specific cysteine and cysteine/serine protease inhibitors, but not by specific serine and aspartic protease inhibitors, with the sole exception of pepstatin A that significantly affects acidic bromelain forms 1 and 2. For all eight protease forms, inhibition is also observed with 1,10-phenanthrolin, a metalloprotease inhibitor. Metal ions (i.e. Mn2+, Mg2+ and Ca2+) showed various effects depending on the protease under consideration, but all of them are totally inhibited in the presence of Zn2+. Mass spectrometry analyses revealed that all forms have a molecular mass of ca. 24 kDa, which is characteristic of enzymes belonging to the papain-like proteases family. Far-UV CD spectra analysis further supported this analysis. Interestingly, secondary structure calculation proves to be highly reproducible for all cysteine proteases of the papain family tested so far (this work; see also Azarkan et al., 2011; Baeyens-Volant et al., 2015) and thus can be used as a test for rapid identification of the classical papain fold. [less ▲]

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See detailA novel protocol for the design of artificial (β/α)8-barrel proteins
Martina, Cristina ULg; Figueroa Yévenes, Maximiliano ULg; Combs, Steven et al

Poster (2016, March)

Designing de novo proteins of more than 100 amino acids is still challenging. The creation of artificial (β/α)8-barrel proteins had only one successful example in literature, thank to use of internal ... [more ▼]

Designing de novo proteins of more than 100 amino acids is still challenging. The creation of artificial (β/α)8-barrel proteins had only one successful example in literature, thank to use of internal spatial symmetry. Here we present a protocol to design de novo (β/α)8-barrel proteins without symmetry restriction. First, the backbone was created in 4 steps: (I) Rosetta ParametricDesign produced an highly symmetric polyalanine scaffold with no loops; (II) Rosetta Fixed-Backbone Design used the previous output to substitute the alanines in all the position; (III) Loops were constructed with Modeller joining the terminus of the secondary structure elements and (IV) RosettaRelax performed relaxation, creating around 4000 different models. 28 backbone models were selected for the next steps of sequence design. To design the final proteins for experimental validation, 10 cycles of Rosetta Design and Relax were performed. In the first cycle only apolar amino acids were allowed in hydrophobic regions; in the next 6 cycles, amino acids were allowed based on the definition of 3 regions: core, boundaries and surface. All the amino acids were allowed in each position in the last 3 cycles. More than 10000 different sequences were created and analyzed in term of amino acid composition, sequence similarity with natural protein, secondary structure prediction, and molecular dynamics simulations. The 30 best candidate sequences have been selected for experimental verification. [less ▲]

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See detailA novel protocol for the design of artificial (β/α)8-barrel proteins
Martina, Cristina ULg; Figueroa Yévenes, Maximiliano ULg; Moretti, Rocco et al

Poster (2016)

The design of protein de novo is an emerging field in biochemistry, where artificial proteins are first designed in silico and then validated experimentally. This research, which rests mainly on our ... [more ▼]

The design of protein de novo is an emerging field in biochemistry, where artificial proteins are first designed in silico and then validated experimentally. This research, which rests mainly on our current understanding of protein structure, function, folding, stability and solubility, contributes to expand our knowledge of proteins in general. Our group has a long tradition in the design of artificial (β/α)8 -barrel proteins (called Octarellins). This fold is extremely interesting because it is widespread in nature (10% of the known proteins contain this fold) and in catalysis (it is present in 5/6 classes of enzyme). Here we present a protocol to design de novo (β/α)8-barrels with the more recent and best performing tools: Rosetta and Modeller (modelling softwares), and GROMACS (molecular dynamic simulations). First, 4000 artificial backbone structures were created with the use of modelling packages Rosetta and Modeller. 54 out of them were selected as targets for the following steps of sequence design and energy minimization (10 cycles), in order to find the best sequence to fit each target. More than 10000 different artificial sequences were created. Selection steps were performed in order to reduce the number of candidates for each target and the best ones were subjected to molecular dynamic simulation. Among this, 5 models were finally chosen for gene synthesis and experimental validation, and are currently being tested for expression in E. coli and preliminary purification. [less ▲]

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See detailBiophysical characterization data of the artificial protein Octarellin V.1 and binding test with its X-ray helpers.
Figueroa Yévenes, Maximiliano ULg; Vandenameele, Julie ULg; Goormaghtigh, Erik et al

in Data in Brief (2016), 8

The artificial protein Octarellin V.1 (http://dx.doi.org/10.1016/j.jsb.2016.05.004[1]) was obtained through a direct evolution process over the de novo designed Octarellin V (http://dx.doi.org/10.1016 ... [more ▼]

The artificial protein Octarellin V.1 (http://dx.doi.org/10.1016/j.jsb.2016.05.004[1]) was obtained through a direct evolution process over the de novo designed Octarellin V (http://dx.doi.org/10.1016/S0022-2836(02)01206-8[2]). The protein has been characterized by circular dichroism and fluorescence techniques, in order to obtain data related to its thermo and chemical stability. Moreover, the data for the secondary structure content studied by circular dichroism and infra red techniques is reported for the Octarellin V and V.1. Two crystallization helpers, nanobodies (http://dx.doi.org/10.1038/nprot.2014.039[3]) and alphaRep (http://dx.doi.org/10.1016/j.jmb.2010.09.048[4]), have been used to create stable complexes. Here we present the data obtained of the binding characterization of the Octarellin V.1 with the crystallization helpers by isothermal titration calorimetry. [less ▲]

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See detailThe role of active site flexible loops in catalysis and of zinc in conformational stability of Bacillus cereus 569/H/9 beta-lactamase.
Montagner, Caroline ULg; Nigen, Michaël; Jacquin, Olivier et al

in Journal of Biological Chemistry (2016), 291(31), 16124-16137

Metallo-beta-lactamases catalyse the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the ... [more ▼]

Metallo-beta-lactamases catalyse the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus beta-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (DeltaG degrees ) of 32 +/- 2 kJ.mol11. For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site and the protein displays a well-organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. 2D NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with DeltaG degrees value of 65 +/- 1.4 kJ.mol11. These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well-defined conformation for both active site loops in order to maintain enzymatic activity. [less ▲]

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See detailThe unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools.
Figueroa Yévenes, Maximiliano ULg; Sleutel, Mike; Vandevenne, Marylène ULg et al

in Journal of structural biology (2016)

Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the ... [more ▼]

Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (alphabetaalpha) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features. [less ▲]

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See detailThe CebE/MsiK Transporter is a Doorway to the Cello-oligosaccharide-mediated Induction of Streptomyces scabies Pathogenicity
Jourdan, Samuel ULg; Francis, Isolde; Kim, Min et al

in Scientific Reports (2016)

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See detailUnravelling the mechanisms of a protein refolding process based on the association of detergents and co-solvents.
Michaux, Catherine; Roussel, Guillaume; Lopes-Rogrigues, M. et al

in Journal of peptide science : an official publication of the European Peptide Society (2016)

A new technique associating the detergent Sodium Dodecyl Sulphate (SDS) and an alcohol-type co-solvent has been set up, showing an unexpected efficiency to refold several types of soluble or membrane ... [more ▼]

A new technique associating the detergent Sodium Dodecyl Sulphate (SDS) and an alcohol-type co-solvent has been set up, showing an unexpected efficiency to refold several types of soluble or membrane proteins. The present contribution deepens the fundamental knowledge on the phenomena underlying this process, considering the refolding of two model peptides featuring the main protein secondary structures: alpha-helix and beta-sheet. Their refolding was monitored by fluorescence and circular dichroism, and it turns out that: (i) 100% recovery of the folded structure is observed for both peptides, (ii) the highest the SDS concentration, the more co-solvent to be added to recover the peptides' native structures, (iii) a high alcohol concentration is required to alter the SDS denaturing properties, (iv) the co-solvent performance relies on its specific lipophilic-hydrophilic balanced character, (v) the size of the micelle formed by the detergent does not enter the process critical parameters, and (vi) increasing the salt concentration up to 1 M NaCl has a beneficial impact on the process efficiency. These mechanistic aspects will help us to improve the method and extend its application. Copyright (c) 2016 European Peptide Society and John Wiley & Sons, Ltd. [less ▲]

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See detailKinetic Study of Laboratory Mutants of NDM-1 Metallo-beta-Lactamase and the Importance of an Isoleucine at Position 35.
Marcoccia, Francesca; Bottoni, Carlo; Sabatini, Alessia et al

in Antimicrobial agents and chemotherapy (2016), 60(4), 2366-72

Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized ... [more ▼]

Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. Thekcat,Km, andkcat/Kmvalues calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, thekcat/Kmof NDM-1(I35S)for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T)and NDM-1(I35S)enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of alpha-helix content in the mutants. [less ▲]

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See detailInfluence of substrate nature and β-lactoglobulin on cleanability after soiling by suspension spraying and drying
Toure, Yetioman ULg; Sindic, Marianne ULg; Dupont-Gillain, C. Christine et al

in Chemical Engineering Science (2015), 134

Glass and stainless steel (StSteel, AISI304-2R), previously cleaned with ethanol (-Eth) or with ethanol and UV–Ozone treatment (-UVO), were soiled with quartz suspensions in water and in a β-lactoglobulin ... [more ▼]

Glass and stainless steel (StSteel, AISI304-2R), previously cleaned with ethanol (-Eth) or with ethanol and UV–Ozone treatment (-UVO), were soiled with quartz suspensions in water and in a β-lactoglobulin (β-LGB) solution, and dried. The cleanability (ease of quartz particle detachment) in water was evaluated using a radial-flow cell. The soiling suspension containing β-LGB was used as such or after heating for 4h at 75°C, which provoked coagulation of about 75% of β-LGB. The substrate–solution interfaces were characterized by X-ray photoelectron spectroscopy (XPS) analysis of conditioned substrates and by contact angle measurements. The substrate surfaces are covered by a layer of organic contaminants which are not removed by pre-cleaning or are adsorbed from the surroundings. The presence of β-LGB in the soiling suspension leads to protein adsorption, but a significant amount of contaminants remains at the surface. For three of the substrates tested (Glass-Eth, Glass-UVO, StSteel-UVO) the increase of cleanability when the soiling suspension contained β-LGB may be explained by lower capillary forces acting upon drying. Capillary forces are proportional to the liquid surface tension and depend in a less important way on substrate contact angle. However the order of cleanability observed for the substrates soiled with a suspension of quartz particles in water (Glass-Eth≅Glass-UVO<StSteel-UVO<StSteel-Eth) and the influence of β-LGB on the cleanability of StSteel-Eth may not be explained only by computed capillary forces. The contact angle may exert a direct influence on droplet spreading and particle–substrate contact. The organic contaminants present on the surfaces, which are often neglected by supposing model solid surfaces, may have a significant influence on cleanability through physico-chemical processes which remain to be appreciated. [less ▲]

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See detailA novel form of ficin from Ficus carica latex: Purification and characterization
Baeyens-Volant, Danielle; Matagne, André ULg; El Mahyaoui, Rachida et al

in Phytochemistry (2015), 117

A novel ficin form, named ficin E, was purified from fig tree latex by a combination of cation-exchange chromatography on SP-Sepharose Fast Flow, Thiopropyl Sepharose 4B and fplc-gel filtration ... [more ▼]

A novel ficin form, named ficin E, was purified from fig tree latex by a combination of cation-exchange chromatography on SP-Sepharose Fast Flow, Thiopropyl Sepharose 4B and fplc-gel filtration chromatography. The new ficin appeared not to be sensitive to thiol derivatization by a polyethylene glycol derivative, allowing its purification. The protease is homogeneous according to PAGE, SDS–PAGE, mass spectrometry, N-terminal micro-sequencing analyses and E-64 active site titration. N-terminal sequencing of the first ten residues has shown high identity with the other known ficin (iso)forms. The molecular weight was found to be (24,294 ± 10) Da by mass spectrometry, a lower value than the apparent molecular weight observed on SDS–PAGE, around 27 kDa. Far-UV CD data revealed a secondary structure content of 22% a-helix and 26% b-sheet. The protein is not glycosylated as shown by carbohydrate analysis. pH and temperature measurements indicated maxima activity at pH 6.0 and 50 °C, respectively. Preliminary pH stability analyses have shown that the protease conserved its compact structure in slightly acidic, neutral and alkaline media but at acidic pH (<3), the formation of some relaxed or molten state was evidenced by 8-anilino-1-naphtalenesulfonic acid binding characteristics. Comparison with the known ficins A, B, C, D1 and D2 (iso)forms revealed that ficin E showed activity profile that looked like ficin A against two chromogenic substrates while it resembled ficins D1 and D2 against three fluorogenic substrates. Enzymatic activity of ficin E was not affected by Mg2+, Ca2+ and Mn2+ at a concentration up to 10 mM. However, the activity was completely suppressed by Zn2+ at a concentration of 1 mM. Inhibitory activity measurements clearly identified the enzyme as a cysteine protease, being unaffected by synthetic (Pefabloc SC, benzamidine) and by natural proteinaceous (aprotinin) serine proteases inhibitors, by aspartic proteases inhibitors (pepstatin A) and by metallo-proteases inhibitors (EDTA, EGTA). Surprisingly, it was well affected by the metallo-protease inhibitor o-phenanthroline. The enzymatic activity was however completely blocked by cysteine proteases inhibitors (E-64, iodoacetamide), by thiol-blocking compounds (HgCl2) and by cysteine/serine proteases inhibitors (TLCK and TPCK). This is a novel ficin form according to peptide mass fingerprint analysis, specific amidase activity, SDS–PAGE and PAGE electrophoretic mobility, N-terminal sequencing and unproneness to thiol pegylation. [less ▲]

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See detailPositive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase.
Bury, Daniel; Dahmane, Ismahene ULg; Derouaux, Adeline ULg et al

in Biochemical pharmacology (2015), 93(2), 141-50

The glycosyltransferases of family 51 (GT51) catalyze the polymerization of lipid II to form linear glycan chains, which, after cross linking by the transpeptidases, form the net-like peptidoglycan ... [more ▼]

The glycosyltransferases of family 51 (GT51) catalyze the polymerization of lipid II to form linear glycan chains, which, after cross linking by the transpeptidases, form the net-like peptidoglycan macromolecule. The essential function of the GT makes it an attractive antimicrobial target; therefore a better understanding of its function and its mechanism of interaction with substrates could help in the design and the development of new antibiotics. In this work, we have used a surface plasmon resonance Biacore((R)) biosensor, based on an amine derivative of moenomycin A immobilized on a sensor chip surface, to investigate the mechanism of binding of substrate analogous inhibitors to the GT. Addition of increasing concentrations of moenomycin A to the Staphylococcus aureus MtgA led to reduced binding of the protein to the sensor chip as expected. Remarkably, in the presence of low concentrations of the most active disaccharide inhibitors, binding of MtgA to immobilized moenomycin A was found to increase; in contrast competition with moenomycin A occurred only at high concentrations. This finding suggests that at low concentrations, the lipid II analogs bind to the acceptor site and induce a cooperative binding of moenomycin A to the donor site. Our results constitute the first indication of the existence of a positive cooperativity between the acceptor and the donor sites of peptidoglycan GTs. In addition, our study indicates that a modification of two residues (L119N and F120S) within the hydrophobic region of MtgA can yield monodisperse forms of the protein with apparently no change in its secondary structure content, but this is at the expense of the enzyme function. [less ▲]

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See detailDetermination of kinetics and the crystal structure of a novel type 2 isopentenyl diphosphate: dimethylallyl diphosphate isomerase from Streptococcus pneumoniae.
de Ruyck, Jerome; Janczak, Matthew W.; Neti, Syam Sundar et al

in Chembiochem : a European journal of chemical biology (2014), 15(10), 1452-1458

Isopentenyl diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI-1 ... [more ▼]

Isopentenyl diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI-1) is a metalloprotein that is found in eukaryotes, whereas the type 2 isoform (IDI-2) is a flavoenzyme found in bacteria that is completely absent from human. IDI-2 from the pathogenic bacterium Streptococcus pneumoniae was recombinantly expressed in Escherichia coli. Steady-state kinetic studies of the enzyme indicated that FMNH2 (KM =0.3 muM) bound before isopentenyl diphosphate (KM =40 muM) in an ordered binding mechanism. An X-ray crystal structure at 1.4 A resolution was obtained for the holoenzyme in the closed conformation with a reduced flavin cofactor and two sulfate ions in the active site. These results helped to further approach the enzymatic mechanism of IDI-2 and, thus, open new possibilities for the rational design of antibacterial compounds against sequence-similar and structure-related pathogens such as Enterococcus faecalis or Staphylococcus aureus. [less ▲]

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See detailClass A β -Lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine
Huynen, Céline ULg; Filée, Patrice; Matagne, André ULg et al

in BioMed Research International (2013), 2013

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications frombasic research to medical applications. This review focuses on the use of class A 𝛽 ... [more ▼]

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications frombasic research to medical applications. This review focuses on the use of class A 𝛽-lactamases as versatile scaffolds to design hybrid enzymes (referred to as 𝛽-lactamase hybrid proteins, BHPs) in which an exogenous peptide, protein or fragment thereof is inserted at various permissive positions.We discuss how BHPs can be specifically designed to create bifunctional proteins, to produce and to characterize proteins that are otherwise difficult to express, to determine the epitope of specific antibodies, to generate antibodies against nonimmunogenic epitopes, and to better understand the structure/function relationship of proteins. [less ▲]

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See detailThe proline-rich motif of the proDer p 3 allergen propeptide is crucial for protease-protease interaction.
Dumez, Marie-Eve ULg; Herman, Julie; Campisi, Vincenzo ULg et al

in PloS one (2013), 8(9), 68014

The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific ... [more ▼]

The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen. [less ▲]

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See detailEnergetics of protein stability at extreme environmental temperatures in bacterial trigger factors
Struvay, Caroline ULg; Negro, Sonia; Matagne, André ULg et al

in Biochemistry (2013), 52

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See detailA Nanobody Binding to Non-amyloidogenic Regions of the Protein Human Lysozyme Enhances Partial Unfolding but Inhibits Amyloid Fibril Formation.
de Genst, EJ; Chan, PH; Pardon, Els et al

in Journal of Physical Chemistry B (2013)

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