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Journal of Bacteriology, September 2008, p. 5855-5861, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00076-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Critical Residues for Cofactor Binding and Catalytic Activity in the Aminoglycoside Resistance Methyltransferase Sgm ^,

Miloje Savic,¹ Tatjana Ilic-Tomic,² Rachel Macmaster,¹ Branka Vasiljevic,² and Graeme L. Conn^1*

Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom,¹ Institute of Molecular Genetics and Genetic Engineering (IMGGE), P.O. Box 23, 11010 Belgrade, Serbia²

Received 15 January 2008/ Accepted 20 June 2008

The 16S rRNA methyltransferase Sgm from "Micromonospora zionensis" confers resistance to aminoglycoside antibiotics by specific modification of the 30S ribosomal A site. Sgm is a member of the FmrO family, distant relatives of the S-adenosyl-L-methionine (SAM)-dependent RNA subfamily of methyltransferase enzymes. Using amino acid conservation across the FmrO family, seven putative key amino acids were selected for mutation to assess their role in forming the SAM cofactor binding pocket or in methyl group transfer. Each mutated residue was found to be essential for Sgm function, as no modified protein could effectively support bacterial growth in liquid media containing gentamicin or methylate 30S subunits in vitro. Using isothermal titration calorimetry, Sgm was found to bind SAM with a K_D (binding constant) of 17.6 µM, and comparable values were obtained for one functional mutant (N179A) and four proteins modified at amino acids predicted to be involved in catalysis in methyl group transfer. In contrast, none of the G135, D156, or D182 Sgm mutants bound the cofactor, confirming their role in creating the SAM binding pocket. These results represent the first functional characterization of any FmrO methyltransferase and may provide a basis for a further structure-function analysis of these aminoglycoside resistance determinants.

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* Corresponding author. Mailing address: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom. Phone: (0161) 3064218. Fax: (0161) 2365201. E-mail: Graeme.L.Conn{at}manchester.ac.uk

Published ahead of print on 27 June 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, September 2008, p. 5855-5861, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00076-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Savic, M., Lovric, J., Tomic, T. I., Vasiljevic, B., Conn, G. L. (2009). Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics. Nucleic Acids Res 37: 5420-5431 [Abstract] [Full Text]  
• Cubrilo, S., Babic, F., Douthwaite, S., Maravic Vlahovicek, G. (2009). The aminoglycoside resistance methyltransferase Sgm impedes RsmF methylation at an adjacent rRNA nucleotide in the ribosomal A site. RNA 15: 1492-1497 [Abstract] [Full Text]