Biomedical subjects

Valérie Guillet

Publications and source records attributed to Valérie Guillet.

7 recordsLinked to original sources

PubMed2005

Further insight into S-adenosylmethionine-dependent methyltransferases: structural characterization of Hma, an enzyme essential for the biosynthesis of oxygenated mycolic acids in Mycobacterium tuberculosis.

Mycolic acids are major and specific components of the cell envelope of Mycobacteria that include Mycobacterium tuberculosis, the causative agent of tuberculosis. Their metabolism is the target of the most efficient antitubercular drug currently used in therapy, and the enzymes that are involved in the production of mycolic acids represent important targets for the development of new drugs effective against multidrug-resistant strains. Among these are the S-adenosylmethionine-dependent methyltransferases (SAM-MTs) that catalyze the introduction of key chemical modifications in defined positions of mycolic acids. Some of these subtle structural variations are known to be crucial for both the virulence of the tubercle bacillus and the permeability of the mycobacterial cell envelope. We report here the structural characterization of the enzyme Hma (MmaA4), a SAM-MT that is unique in catalyzing the introduction of a methyl branch together with an adjacent hydroxyl group essential for the formation of both keto- and methoxymycolates in M. tuberculosis. Despite the high propensity of Hma to proteolytic degradation, the enzyme was produced and crystallized, and its three-dimensional structure in the apoform and in complex with S-adenosylmethionine was solved to about 2 A. Thestructuresshowtheimportantroleplayedbythemodificationsfound within mycolic acid SAM-MTs, especially thealpha2-alpha3 motif and the chemical environment of the active site. Essential information with respect to cofactor and substrate binding, selectivity and specificity, and about the mechanism of catalytic reaction were derived.

Amino Acid Sequence↗

PubMed2004

Advanced method using microwaves and solid-phase microextraction coupled with gas chromatography-mass spectrometry for the determination of pyrethroid residues in strawberries.

A microwave-assisted desorption method was developed and coupled with solid-phase microextraction and GC-MS for the analysis of pyrethroid residues in strawberries. In the first step, pyrethroid analytes were desorbed from the whole fruits in an aqueous acetonitrile solution at 50% under microwave assistance, so preventing these compounds to be captured with strong matrix effects by endogenous constituents. Then, the 100 microm poly(dimethylsiloxane)-coated fibre was exposed for 30 min in the obtained extracting solution. Calibration curves, realised from blank strawberries spiked at different concentrations with standards, showed a linear range between 1 microg/kg and 250 microg/kg with r2 > 0.992 and variation coefficients below 15%. Limits of detection and quantitation were found lower than 14 microg/kg and 40 microg/kg, respectively. Observed analysis results by using this method and relative to field incurred strawberry samples were also compared to those obtained by two accredited trading laboratories using traditional methods.

Adsorption↗

PubMed2004

Crystal structure of leucotoxin S component: new insight into the Staphylococcal beta-barrel pore-forming toxins.

Staphylococcal leucocidins and gamma-hemolysins (leucotoxins) are bi-component toxins that form lytic transmembrane pores. Their cytotoxic activities require the synergistic association of a class S component and a class F component, produced as water-soluble monomers that form hetero-oligomeric membrane-associated complexes. Strains that produce the Panton-Valentine leucocidin are clinically associated with cutaneous lesions and community-acquired pneumonia. In a previous study, we determined the crystal structure of the F monomer from the Panton-Valentine leucocidin. To derive information on the second component of the leucotoxins, the x-ray structure of the S protein from the Panton-Valentine leucocidin was solved to 2.0 angstrom resolution using a tetragonal crystal form that contains eight molecules in the asymmetric unit. The structure demonstrates the different conformation of the domain involved in membrane contacts and illustrates sequence and tertiary structure variabilities of the pore-forming leucotoxins. Mutagenesis studies at a key surface residue (Thr-28) further support the important role played by these microheterogeneities for the assembly of the bipartite leucotoxins.

Amino Acid Sequence↗

PubMed2004

Crystallization and preliminary crystallographic data of a leucotoxin S component from Staphylococcus aureus.

Class S proteins of staphylococcal bicomponent pore-forming leucotoxins play an important role in membrane targetting and cell specificity. Wild-type and recombinant S components of the Panton-Valentine leucocidin (LukS-PV) were expressed in Staphylococcus aureus and Escherichia coli, respectively, and purified. Both proteins were crystallized in two crystal forms with Jeffamine M-600 as the precipitant at 285 K using the hanging-drop vapour-diffusion method and seeding techniques. Crystals belong to space group P2 (or P2(1)) and P4(1) (or P4(3)), with unit-cell parameters a = 72.3, b = 95.1, c = 108.1 A, beta = 106.4 degrees and a = b = 94.8, c = 306.2 A, respectively. A full set of X-ray diffraction data was collected to 2.1 A from a single tetragonal crystal of the wild-type protein at 100 K.

Crystallography, X-Ray↗

PubMed2002

Crystallographic and biochemical studies of DivK reveal novel features of an essential response regulator in Caulobacter crescentus.

DivK is an essential response regulator in the Gram-negative bacterium Caulobacter crescentus and functions in a complex phosphorelay system that precisely controls the sequence of developmental events during the cell division cycle. Structure determinations of this single domain response regulator at different pH values demonstrated that the five-stranded alpha/beta fold of the DivK protein is fully defined only at acidic pH. The crystal structures of the apoprotein and of metal-bound DivK complexes at higher pH values revealed a synergistic pH- and cation binding-induced flexibility of the beta4-alpha4 loop and of the alpha4 helix. This motion increases the solvent accessibility of the single cysteine residue in the protein. Solution state studies demonstrated a 200-fold pH-dependent increase in the affinity of manganese for the protein between pH 6.0 and 8.5 that seems to involve deprotonation of an acido-basic couple. Taken together, these results suggest that flexibility of critical regions of the protein, ionization of the cysteine 99 residue and improved K(D) values for the catalytic metal ion are coupled events. We propose that the molecular events observed in the isolated protein may be required for DivK activation and that they may be achieved in vivo through the specific protein-protein interactions between the response regulator and its cognate kinases.

Amino Acid Sequence↗

PubMed2002

Characterization and crystallization of DivK, an essential response regulator for cell division and differentiation in Caulobacter crescentus.

DivK is an essential response regulator involved in the complex signal transduction network required for cell division and cell differentiation in Caulobacter crescentus. Small-angle X-ray scattering analysis was valuable for obtaining single crystals of the DivK recombinant protein. These crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 37.2, b = 40.5, c = 67.1 A and diffract beyond 1.6 A on a synchrotron beamline.

Bacterial Proteins↗

PubMed2002

Structure of the imipenem-hydrolyzing class A beta-lactamase SME-1 from Serratia marcescens.

The structure of the beta-lactamase SME-1 from Serratia marcescens, a class A enzyme characterized by its significant activity against imipenem, has been determined to 2.13 A resolution. The overall structure of SME-1 is similar to that of other class A beta-lactamases. In the active-site cavity, most of the residues found in SME-1 are conserved among class A beta-lactamases, except at positions 104, 105 and 237, where a tyrosine, a histidine and a serine are found, respectively, and at position 238, which is occupied by a cysteine forming a disulfide bridge with the other cysteine residue located at position 69. The crucial role played by this disulfide bridge in SME-1 was confirmed by site-directed mutagenesis of Cys69 to Ala, which resulted in a mutant unable to confer resistance to imipenem and all other beta-lactam antibiotics tested. Another striking structural feature found in SME-1 was the short distance separating the side chains of the active serine residue at position 70 and the strictly conserved glutamate at position 166, which is up to 1.4 A shorter in SME-1 compared with other class A beta-lactamases. Consequently, the SME-1 structure cannot accommodate the essential catalytic water molecule found between Ser70 and Glu166 in the other class A beta-lactamases described so far, suggesting that a significant conformational change may be necessary in SME-1 to properly position the hydrolytic water molecule involved in the hydrolysis of the acyl-enzyme intermediate.

Alanine↗