Critical residues for cofactor binding and catalytic activity in the aminoglycoside resistance methyltransferase Sgm
Апстракт
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 mu M, 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 structurefunction analysis of these aminoglycoside resistance determinants.
Извор:
Journal of Bacteriology, 2008, 190, 17, 5855-5861Издавач:
- Amer Soc Microbiology, Washington
Финансирање / пројекти:
- Wellcome Trust [078374]
- Експресија и регулација фармацеутски значајних гена у микроорганизмима (RS-MESTD-MPN2006-2010-143056)
DOI: 10.1128/JB.00076-08
ISSN: 0021-9193
PubMed: 18586937
WoS: 000258695400013
Scopus: 2-s2.0-50249152954
Институција/група
Institut za molekularnu genetiku i genetičko inženjerstvoTY - JOUR AU - Savić, Miloje AU - Ilić-Tomić, Tatjana AU - Macmaster, Rachel AU - Vasiljević, Branka AU - Conn, Graeme L. PY - 2008 UR - https://imagine.imgge.bg.ac.rs/handle/123456789/328 AB - 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 mu M, 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 structurefunction analysis of these aminoglycoside resistance determinants. PB - Amer Soc Microbiology, Washington T2 - Journal of Bacteriology T1 - Critical residues for cofactor binding and catalytic activity in the aminoglycoside resistance methyltransferase Sgm EP - 5861 IS - 17 SP - 5855 VL - 190 DO - 10.1128/JB.00076-08 ER -
@article{ author = "Savić, Miloje and Ilić-Tomić, Tatjana and Macmaster, Rachel and Vasiljević, Branka and Conn, Graeme L.", year = "2008", abstract = "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 mu M, 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 structurefunction analysis of these aminoglycoside resistance determinants.", publisher = "Amer Soc Microbiology, Washington", journal = "Journal of Bacteriology", title = "Critical residues for cofactor binding and catalytic activity in the aminoglycoside resistance methyltransferase Sgm", pages = "5861-5855", number = "17", volume = "190", doi = "10.1128/JB.00076-08" }
Savić, M., Ilić-Tomić, T., Macmaster, R., Vasiljević, B.,& Conn, G. L.. (2008). Critical residues for cofactor binding and catalytic activity in the aminoglycoside resistance methyltransferase Sgm. in Journal of Bacteriology Amer Soc Microbiology, Washington., 190(17), 5855-5861. https://doi.org/10.1128/JB.00076-08
Savić M, Ilić-Tomić T, Macmaster R, Vasiljević B, Conn GL. Critical residues for cofactor binding and catalytic activity in the aminoglycoside resistance methyltransferase Sgm. in Journal of Bacteriology. 2008;190(17):5855-5861. doi:10.1128/JB.00076-08 .
Savić, Miloje, Ilić-Tomić, Tatjana, Macmaster, Rachel, Vasiljević, Branka, Conn, Graeme L., "Critical residues for cofactor binding and catalytic activity in the aminoglycoside resistance methyltransferase Sgm" in Journal of Bacteriology, 190, no. 17 (2008):5855-5861, https://doi.org/10.1128/JB.00076-08 . .