André Matagne

Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae.

ABSTRACT Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII β-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those β-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 β-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of β-lactamases.

Contribution of Mutant Analysis to the Understanding of Enzyme Catalysis: The Case of Class a Beta-Lactamases

Class A beta-lactamases represent a family of well studied enzymes. They are responsible for many antibiotic resistance phenomena and thus for numerous failures in clinical chemotherapy. Despite the facts that five structures are known at high resolution and that detailed analyses of enzymes modified by site-directed mutagenesis have been performed, their exact catalytic mechanism remains controversial. This review attempts to summarize and to discuss the many available data.

Mutational Analysis of VIM-2 Reveals an Essential Determinant for Metallo-β-Lactamase Stability and Folding

ABSTRACT Metallo-β-lactamase (MBL)-producing bacteria are emerging worldwide and represent a formidable threat to the efficacy of relevant β-lactams, including carbapenems, expanded-spectrum cephalosporins, and β-lactamase inactivator/β-lactam combinations. VIM-2 is currently the most widespread MBL and represents a primary target for MBL inhibitor research, the clinical need for which is expected to further increase in the future. Using a saturation mutagenesis approach, we probed the importance of four residues (Phe-61, Ala-64, Tyr-67, and Trp-87) located close to the VIM-2 active site and putatively relevant to the enzyme activity based on structural knowledge of the enzyme and on structure-activity relationships of the subclass B1 MBLs. The ampicillin MIC values shown by the various mutants were affected very differently depending on the randomized amino acid position. Position 64 appeared to be rather tolerant to substitution, and kinetic studies showed that the A64W mutation did not significantly affect substrate hydrolysis or binding, representing an important difference from IMP-type enzymes. Phe-61 and Tyr-67 could be replaced with several amino acids without the ampicillin MIC being significantly affected, but in contrast, Trp-87 was found to be critical for ampicillin resistance. Further kinetic and biochemical analyses of W87A and W87F variants showed that this residue is apparently important for the structure and proper folding of the enzyme but, surprisingly, not for its catalytic activity. These data support the critical role of residue 87 in the stability and folding of VIM-2 and might have strong implications for MBL inhibitor design, as this residue would represent an ideal target for interaction with small molecules.

Kinetic study of interaction between BRL 42715, beta-lactamases, and D-alanyl-D-alanine peptidases.

A detailed kinetic study of the interactions between BRL 42715, a beta-lactamase-inhibiting penem, and various beta-lactamases (EC 3.5.2.6) and D-alanyl-D-alanine peptidases (DD-peptidases, EC 3.4.16.4) is presented. The compound was a very efficient inactivator of all active-site serine beta-lactamases but was hydrolyzed by the class B, Zn(2+)-containing enzymes, with very different kcat values. Inactivation of the Streptomyces sp. strain R61 extracellular DD-peptidase was not observed, and the Actinomadura sp. strain R39 DD-peptidase exhibited a low level of sensitivity to the compound.

A Nanobody Binding to Non-amyloidogenic Regions of the Protein Human Lysozyme Enhances Partial Unfolding but Inhibits Amyloid Fibril Formation.

We report the effects of the interaction of two camelid antibody fragments, generally called nanobodies, namely cAb-HuL5 and a stabilized and more aggregation-resistant variant cAb-HuL5G obtained by protein engineering, on the properties of two amyloidogenic variants of human lysozyme, I56T and D67H, whose deposition in vital organs including the liver, kidney, and spleen is associated with a familial non-neuropathic systemic amyloidosis. Both NMR spectroscopy and X-ray crystallographic studies reveal that cAb-HuL5 binds to the α-domain, one of the two lobes of the native lysozyme structure. The binding of cAb-HuL5/cAb-HuL5G strongly inhibits fibril formation by the amyloidogenic variants; it does not, however, suppress the locally transient cooperative unfolding transitions, characteristic of these variants, in which the β-domain and the C-helix unfold and which represents key early intermediate species in the formation of amyloid fibrils. Therefore, unlike two other nanobodies previously described, cAb-HuL5/cAb-HuL5G does not inhibit fibril formation via the restoration of the global cooperativity of the native structure of the lysozyme variants to that characteristic of the wild-type protein. Instead, it inhibits a subsequent step in the assembly of the fibrils, involving the unfolding and structural reorganization of the α-domain. These results show that nanobodies can protect against the formation of pathogenic aggregates at different stages in the structural transition of a protein from the soluble native state into amyloid fibrils, illustrating their value as structural probes to study the molecular mechanisms of amyloid fibril formation. Combined with their amenability to protein engineering techniques to improve their stability and solubility, these findings support the suggestion that nanobodies can potentially be developed as therapeutics to combat protein misfolding diseases.

Three factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys 70

The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase.

Stepwise Adaptations to Low Temperature as Revealed by Multiple Mutants of Psychrophilic α-Amylase from Antarctic Bacterium

Background: Cold-adapted enzymes remain catalytically active at low temperatures. Results: Mutants of a cold-adapted α-amylase stabilized by engineered weak interactions and a disulfide bond have lost the kinetic optimization to low temperatures. Conclusion: The disappearance of stabilizing interactions in psychrophilic enzymes increases the dynamics of active site residues at low temperature, leading to a higher activity. Significance: An experimental support to the activity-stability relationships. The mutants Mut5 and Mut5CC from a psychrophilic α-amylase bear representative stabilizing interactions found in the heat-stable porcine pancreatic α-amylase but lacking in the cold-active enzyme from an Antarctic bacterium. From an evolutionary perspective, these mutants can be regarded as structural intermediates between the psychrophilic and the mesophilic enzymes. We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: compactness; kinetically driven stability; thermodynamic stability; resistance toward chemical denaturation, and the kinetics of unfolding/refolding. Concomitantly to this improved stability, both mutants have lost the kinetic optimization to low temperature activity displayed by the parent psychrophilic enzyme. These results provide strong experimental support to the hypothesis assuming that the disappearance of stabilizing interactions in psychrophilic enzymes increases the amplitude of concerted motions required by catalysis and the dynamics of active site residues at low temperature, leading to a higher activity.

The zinc center Influences the redox and thermodynamic properties of Escherichia coli thioredoxin 2

Thioredoxins are small, ubiquitous redox enzymes that reduce protein disulfide bonds by using a pair of cysteine residues present in a strictly conserved WCGPC catalytic motif. The Escherichia coli cytoplasm contains two thioredoxins, Trx1 and Trx2. Trx2 is special because it is induced under oxidative stress conditions and it has an additional N-terminal zinc-binding domain. We have determined the redox potential of Trx2, the pK(a) of the active site nucleophilic cysteine, as well as the stability of the oxidized and reduced form of the protein. Trx2 is more oxidizing than Trx1 (-221 mV versus -284 mV, respectively), which is in good agreement with the decreased value of the pK(a) of the nucleophilic cysteine (5.1 versus 7.1, respectively). The difference in stability between the oxidized and reduced forms of an oxidoreductase is the driving force to reduce substrate proteins. This difference is smaller for Trx2 (DeltaDeltaG degrees(H2O)=9 kJ/mol and DeltaT(m)=7. 4 degrees C) than for Trx1 (DeltaDeltaG degrees(H2O)=15 kJ/mol and DeltaT(m)=13 degrees C). Altogether, our data indicate that Trx2 is a significantly less reducing enzyme than Trx1, which suggests that Trx2 has a distinctive function. We disrupted the zinc center by mutating the four Zn(2+)-binding cysteines to serine. This mutant has a more reducing redox potential (-254 mV) and the pK(a) of its nucleophilic cysteine shifts from 5.1 to 7.1. The removal of Zn(2+) also decreases the overall stability of the reduced and oxidized forms by 3.2 kJ/mol and 5.8 kJ/mol, respectively. In conclusion, our data show that the Zn(2+)-center of Trx2 fine-tunes the properties of this unique thioredoxin.

Positively Cooperative Binding of Zinc Ions to Bacillus cereus 569/H/9 β-Lactamase II Suggests that the Binuclear Enzyme Is the Only Relevant Form for Catalysis

Metallo-beta-lactamases catalyze the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. While enzymes belonging to subclass B1 have been shown to display maximum activity as dizinc species, the actual metal-to-protein stoichiometry and the affinity for zinc are not clear. We have further investigated the process of metal binding to the beta-lactamase II from Bacillus cereus 569/H/9 (known as BcII). Zinc binding was monitored using complementary biophysical techniques, including circular dichroism in the far-UV, enzymatic activity measurements, competition with a chromophoric chelator, mass spectrometry, and nuclear magnetic resonance. Most noticeably, mass spectrometry and nuclear magnetic resonance experiments, together with catalytic activity measurements, demonstrate that two zinc ions bind cooperatively to the enzyme active site (with K(1)/K(2)> or =5) and, hence, that catalysis is associated with the dizinc enzyme species only. Furthermore, competitive experiments with the chromophoric chelator Mag-Fura-2 indicates K(2)<80 nM. This contrasts with cadmium binding, which is clearly a noncooperative process with the mono form being the only species significantly populated in the presence of 1 molar equivalent of Cd(II). Interestingly, optical measurements reveal that although the apo and dizinc species exhibit undistinguishable tertiary structural organizations, the metal-depleted enzyme shows a significant decrease in its alpha-helical content, presumably associated with enhanced flexibility.

Quantitative analysis of the stabilization by substrate of Staphylococcus aureus PC1 β-lactamase

BACKGROUND The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 beta-lactamase, at temperatures above the melting point of the enzyme. RESULTS Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K(m) values, significant stabilization was observed with all tested compounds. A combination of unfolding kinetic measurements and enzymatic studies, both under steady-state and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis and enzyme denaturation) to be evaluated. In addition, molecular modelling studies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme. CONCLUSIONS Our analysis indicates that the enzyme is substantially stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri-Michaelis complex (ES) and acyl-enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem.