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Antimicrobial Agents and Chemotherapy, December 2005, p. 5112-5118, Vol. 49, No. 12
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.12.5112-5118.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Analysis of the Contribution of Individual Substituents in 4,6-Aminoglycoside-Ribosome Interaction

Sven N. Hobbie,¹ Peter Pfister,¹ Christian Brüll,¹ Eric Westhof,² and Erik C. Böttger^1*

Institut für Medizinische Mikrobiologie, Universität Zürich, Gloriastr. 30/32, CH-8006 Zürich, Switzerland,¹ Institut de Biologie Moléculaire et Cellulaire du CNRS, Modélisation et Simulations des Acides Nucléiques UPR 9002, Université Louis Pasteur, 15 rue René Descartes, F-67084 Strasbourg Cedex, France²

Received 12 May 2005/ Returned for modification 2 August 2005/ Accepted 12 September 2005

The 4,6-disubstituted 2-deoxystreptamines interfere with protein biosynthesis by specifically targeting the ribosomal A site. These drugs show subtle variations in the chemical groups of rings I, II, and III. In the present study we used site-directed mutagenesis to generate mutant strains of Mycobacterium smegmatis mc²155 SMR5 rrnB with mutations in its single rRNA allele, rrnA. This genetic procedure gives rise to strains carrying homogeneous populations of mutant ribosomes and was used to study the contribution of individual chemical substituents to the binding of 4,6-disubstituted aminoglycosides. X-ray crystal structures of geneticin and tobramycin complexed to oligonucleotides containing the minimal bacterial ribosomal A site were used for interpretation of MICs determined for a panel of 4,6-aminoglycosides, including tobramycin, kanamycin A, kanamycin B, amikacin, gentamicin, and geneticin. Surprisingly, the considerable differences present within ring III did not seem to alter the interaction of the drug with the ribosome, as determined by site-directed mutagenesis of the A site. In contrast, subtle variations in ring I significantly influenced binding: (i) a 4'-hydroxyl moiety participates in the proper drug target interaction; and (ii) a 2'-amino group contributes an additional positive charge to ring I, making the drug less susceptible to any kind of sequence alteration within the decoding site, most notably, to conformational changes induced by transversion of U1495 to 1495A. The 4-amino-2-hydroxyl-1-oxobutyl extension at position 1 of ring II of amikacin provides an additional anchor and renders amikacin less dependent on the structural conformation of nucleotide U1406 compared to the dependencies of other kanamycins. Overall, the set of interactions forming the complex between drug substituents and nucleotides of the A site constitutes a network in which the interactions can partly compensate for each other when they are disrupted.

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* Corresponding author. Mailing address: Institut für Medizinische Mikrobiologie, Universität Zürich, Gloriastr. 30/32, CH-8006 Zürich, Switzerland. Phone: 41-(0)44-634 26 60/61. Fax: 41-(0)44-634 49 06. E-mail: boettger{at}immv.unizh.ch.

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Antimicrobial Agents and Chemotherapy, December 2005, p. 5112-5118, Vol. 49, No. 12
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.12.5112-5118.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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