Nucleic Acids Research Advance Access originally published online on July 9, 2009
Nucleic Acids Research 2009 37(16):5420-5431; doi:10.1093/nar/gkp575
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Nucleic Acids Research, 2009, Vol. 37, No. 16 5420-5431
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics
1,*1Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK, 2Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, P. O. Box 23, 11010 Beograd, Serbia and 3Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta GA 30322, USA
*To whom correspondence should be addressed. Tel: +1 404 727 5965; Fax: +1 404 727 2738; Email: gconn{at}emory.edu
Correspondence may also be addressed to Josip Lovri. Tel: +44 161 306 4476; Fax: +44 161 306 5201; Email: josip.lovric{at}manchester.ac.uk
Received April 7, 2009. Revised June 19, 2009. Accepted June 22, 2009.
The 16S ribosomal RNA methyltransferase enzymes that modify nucleosides in the drug binding site to provide self-resistance in aminoglycoside-producing micro-organisms have been proposed to comprise two distinct groups of S-adenosyl-L-methionine (SAM)-dependent RNA enzymes, namely the Kgm and Kam families. Here, the nucleoside methylation sites for three Kgm family methyltransferases, Sgm from Micromonospora zionensis, GrmA from Micromonospora echinospora and Krm from Frankia sp. Ccl3, were experimentally determined as G1405 by MALDI-ToF mass spectrometry. These results significantly extend the list of securely characterized G1405 modifying enzymes and experimentally validate their grouping into a single enzyme family. Heterologous expression of the KamB methyltransferase from Streptoalloteichus tenebrarius experimentally confirmed the requirement for an additional 60 amino acids on the deduced KamB N-terminus to produce an active methyltransferase acting at A1408, as previously suggested by an in silico analysis. Finally, the modifications at G1405 and A1408, were shown to confer partially overlapping but distinct resistance profiles in Escherichia coli. Collectively, these data provide a more secure and systematic basis for classification of new aminoglycoside resistance methyltransferases from producers and pathogenic bacteria on the basis of their sequences and resistance profiles.