HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 37, 25332-25339, September 12, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/37/25332    most recent M800418200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Jacquet, E. Articles by Chesneau, O. PubMed PubMed Citation Articles by Jacquet, E. Articles by Chesneau, O. Social Bookmarking                      What's this?

ATP Hydrolysis and Pristinamycin IIA Inhibition of the Staphylococcus aureus Vga(A), a Dual ABC Protein Involved in Streptogramin A Resistance^*

Eric Jacquet¹², Jean-Marie Girard¹³⁴, Odile Ramaen¹³⁵, Olivier Pamlard, Hélène Lévaique, Jean-Michel Betton, Elie Dassa^¶, and Olivier Chesneau^¶

From the Institut de Chimie des Substances Naturelles, CNRS UPR 2301, avenue de la Terrasse, Gif-sur-Yvette 91190 and the Unité de Biochimie Structurale and ^¶Unité des Membranes Bactériennes, Institut Pasteur, CNRS URA 2172, 25 rue du Docteur Roux, Paris 75724 Cedex 15, 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 µM, 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.

Received for publication, January 16, 2008 , and in revised form, June 12, 2008.

^* This work was supported in part by Institut Pasteur Grant PTR 55 (to O. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ These authors contributed equally to this work.

³ Supported by grants from the Institut de Chimie des Substances Naturelles.

⁴ Present address: The Hospital for Sick Children, Program in Genetic and Genomic Biology, MaRS Discovery District, 101 College St., Ste. 15-401J, Toronto, Ontario M5G 1L7, Canada.

⁵ Present address: Laboratoire d'Enzymologie et Biologie Structurales, CNRS, avenue de la Terrasse, Gif-sur-Yvette 91190, France.

² To whom correspondence should be addressed. Tel.: 33-1-69-82-35-79; Fax: 33-1-69-82-36-48; E-mail: Eric.Jacquet{at}icsn.cnrs-gif.fr.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?