ATP hydrolysis and pristinamycin IIA inhibition of the Staphylococcus aureus Vga(A), a dual ABC protein involved in streptogramin A resistance.

J Biol Chem 2008 Sep 18;283(37):25332-9. Epub 2008 Jun 18.

Institut de Chimie des Substances Naturelles, avenue de la Terrasse, Gif-sur-Yvette, France.

In Gram-positive bacteria, a large subfamily of dual ATP-binding cassette proteins confers acquired or intrinsic resistance to macrolide, lincosamide, and streptogramin antibiotics by a far from well understood mechanism. Here, we report the first biochemical characterization of one such protein, Vga(A), which is involved in streptogramin A (SgA) resistance among staphylococci. Vga(A) is composed of two nucleotide-binding domains (NBDs), separated by a charged linker, with a C-terminal extension and without identified transmembrane domains. Highly purified Vga(A) displays a strong ATPase activity (K(m) = 78 mum, V(m) = 6.8 min(-1)) that was hardly inhibited by orthovanadate. Using mutants of the conserved catalytic glutamate residues, the two NBDs of Vga(A) were shown to contribute unequally to the total ATPase activity, the mutation at NBD2 being more detrimental than the other. ATPase activity of both catalytic sites was essential for Vga(A) biological function because each single Glu mutant was unable to confer SgA resistance in the staphylococcal host. Of great interest, Vga(A) ATPase was specifically inhibited in a non-competitive manner by the SgA substrate, pristinamycin IIA (PIIA). A deletion of the last 18 amino acids of Vga(A) slightly affected the ATPase activity without modifying the PIIA inhibition values. In contrast, this deletion reduced 4-fold the levels of SgA resistance. Altogether, our results suggest a role for the C terminus in regulation of the SgA antibiotic resistance mechanism conferred by Vga(A) and demonstrate that this dual ATP-binding cassette protein interacts directly and specifically with PIIA, its cognate substrate.

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http://dx.doi.org/10.1074/jbc.M800418200DOI Listing
September 2008
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