Comparative study of mature and zymogen mite cysteine protease stability and pH unfolding.

in Biochimica et Biophysica Acta (2010), 1800(9), 937-945

BACKGROUND: Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes. METHODS: To ... [more ▼]

BACKGROUND: Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes. METHODS: To have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1. RESULTS: We showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen. GENERAL SIGNIFICANCE: Finally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis. [less ▲]

Effect of polyQ insertions on the structural, thermodynamic and aggregating properties of the beta-lactamase from B. Licheniformis 749/C (BlaP)

Poster (2009, April 28)

Critical role of tryptophan 154 for the activity and stability of class D beta-lactamases.

in Biochemistry (2009), 48(47), 11252-63

The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of ... [more ▼]

The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of catalysis. Evidence is presented that the interaction between the indole group of Trp154 and the carboxylated lysine is essential for the stability of the posttranslationally modified Lys70. Substitution of Trp154 by Gly, Ala, or Phe yielded noncarboxylated enzymes which displayed poor catalytic efficiencies and reduced stability when compared to the wild-type OXA-10. The W154H mutant was partially carboxylated. In addition, the maximum values of k(cat) and k(cat)/K(M) were shifted toward pH 7, indicating that the carboxylation state of Lys70 is dependent on the protonation level of the histidine. A comparison of the three-dimensional structures of the different proteins also indicated that the Trp154 mutations did not modify the overall structures of OXA-10 but induced an increased flexibility of the Omega-loop in the active site. Finally, the deacylation-impaired W154A mutant was used to determine the structure of the acyl-enzyme complex with benzylpenicillin. These results indicate a role of the Lys70 carboxylation during the deacylation step and emphasize the importance of Trp154 for the ideal positioning of active site residues leading to an optimum activity. [less ▲]

The in-vitro antimicrobial activity of some medicinal plants against beta-lactam-resistant bacteria

in J Infect Dev Ctries (2009), 3(9), 671-80

BACKGROUND: In effort to identify novel bacterial agents, this study was initiated to evaluate the antimicrobial properties of 17 crude extracts from 12 medicinal plants against beta-lactam-resistant ... [more ▼]

BACKGROUND: In effort to identify novel bacterial agents, this study was initiated to evaluate the antimicrobial properties of 17 crude extracts from 12 medicinal plants against beta-lactam-resistant bacteria. METHODOLOGY: The antimicrobial activities of plant extracts were evaluated against clinically proved beta-lactam-resistant bacteria (Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Serratia marcescens, Acinetobacter baumannii, Staphylococcus aureus and Enterococcus sp.) and reference strains of bacteria (Escherichia coli ATCC 35218, Enterobacter aerogenes ATCC 29751, E. aerogenes ATCC 13048, Pseudomonas aeruginosa ATCC 27853 and Enterococcus hirae ATCC 9790) by using disc-diffusion and agar-dilution assays. RESULTS: The crude plant extracts demonstrated broad spectrum activity against all bacteria tested with inhibition zones in the range of 8-30 mm. The minimal inhibitory concentration (MIC) values of different plant extracts against the tested bacteria were found to range from <or= 0.3 to >or= 10 mg ml(-1). The most active plant extracts were from Dortenia picta and Bridelia micrantha (MIC: 1.25-10 mg ml(-1)) on beta-lactam-resistant Gram-negative bacilli and the extracts from B. micrantha, Mallotus oppositifolius, Garcinia lucida, Garcinia. kola, Campylospermum densiflorum (leaves) and C. zenkeri (root) on beta-lactam-resistant Gram-positive cocci (MIC: <or= 0.3-5 mg ml(-1)). CONCLUSION: Of the 17 plant extracts studied, seven showed good antimicrobial activity against the tested bacteria. The stem bark of B. micrantha and the leaves of D. picta were most active towards beta-lactamase producing Gram-negative bacilli. This study shows that medicinal plants could be sources of compounds which can be used to fight against beta-lactam resistant bacteria. [less ▲]

Chimeric proteins as models to study the mechanism of aggregation associated with polyglutamine expansions

Conference (2008, September)

Creation of chimeric proteins as models to study the mechanism of aggregation associated with polyglutamine expansions

Poster (2008, July 20)

Characterization of the cattle serum antibody responses against TEM beta-lactamase and the nonimmunogenic Escherichia coli heat-stable enterotoxin (STaI)

in FEMS Immunology & Medical Microbiology (2008), 54(3), 319-329

In order to test the use of a subunit recombinant vaccine for its capacity to induce antibodies against the nonimmunogenic heat-stable enterotoxin STa from Escherichia coli and the TEM-1 beta-lactamase ... [more ▼]

In order to test the use of a subunit recombinant vaccine for its capacity to induce antibodies against the nonimmunogenic heat-stable enterotoxin STa from Escherichia coli and the TEM-1 beta-lactamase, cattle were immunized with a hybrid protein created by insertion of the STa sequence at position 197 of the TEM-1 beta-lactamase. Specific anti-STa IgG and IgG1 antibodies were detected at low levels, while no IgG2 antibodies were detected. In contrast, high levels of the different anti-TEM IgG subtypes were detected in cattle sera. In addition, beta-lactamase activity was inhibited by the sera. The presence of antibodies against STa and TEM-1 beta-lactamase was assessed in sera from 366 cattle taken from the field. No significant level of IgGs against the toxin or the TEM-1 was detected. A comparison of the antibody level between the immunized and the nonimmunized animals clearly demonstrated that STa was not able to induce a significant level of antibodies in the vaccinated animals. In contrast, a strong antibody response against TEM-1 beta-lactamase was demonstrated. [less ▲]

Creating hybrid proteins by insertion of exogenous peptides into permissive sites of a class A beta-lactamase

in FEBS Journal (2008), 275

Mutational analysis of the zinc- and substrate-binding sites in the CphA metallo-beta-lactamase from Aeromonas hydrophila.

in Biochemical Journal (2008), 414(1), 151-9

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate ... [more ▼]

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His(118), Asp(120), His(196) and His(263)) and in substrate specificity (Val(67), Thr(157), Lys(224) and Lys(226)), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp(120) and His(263), and thus is located in the 'cysteine' zinc-binding site. His(118) and His(196) residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val(67) plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val(67) also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys(224) in the binding of substrate. Lys(226) is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding. [less ▲]

Relationship between propeptide pH unfolding and inhibitory ability during ProDer p 1 activation mechanism

in Journal of Molecular Biology (2007), 374(1), 170-185

The major allergen Der p1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) produced as an inactive precursor and associated with allergic diseases. The ... [more ▼]

The major allergen Der p1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) produced as an inactive precursor and associated with allergic diseases. The propeptide of Der p I exhibits a specific fold that makes it unique in the CA1 propeptide family. In this study, we investigated the activation steps involved in the maturation of the recombinant protease Der p 1 expressed in Pichia pastoris and the interaction of the full-length and truncated soluble propepticles with their parent enzyme in terms of activity inhibition and BIAcore interaction analysis. According to our results, the activation of protease Der p 1 is a multistep mechanism that is characterized by at least two intermediates. The propeptide strongly inhibits unglycosylated and glycosylated recombinant Der p 1 (K-D = 7 nM) at neutral pH. This inhibition is pH dependent. It decreases from pH 7 to pH 4 and can be related to conformational changes of the propepticle characterized by an increase of its flexibility and formation of a molten globule state. Our results indicate that activation of the zymogen at pH 4 is a compromise between activity preservation and propeptide unfolding. (c) 2007 Elsevier Ltd. All rights reserved. [less ▲]