TY  - JOUR
AU  - Morić, Ivana
AU  - Jeremić, Sanja
AU  - Savić, Miloje
AU  - Ilić-Tomić, Tatjana
AU  - Conn, Graeme L.
AU  - Vasiljević, Branka
PY  - 2009
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/374
AB  - The mechanism of resistance to aminoglycosides based on methylation of their target, 16S rRNA, was until recently described only in antibiotic producing microorganisms. However, equivalent methyltransferases have now also been identified among numerous clinical Gram-negative pathogenic isolates. We have cloned, expressed, and purified GrmA, the aminoglycoside-resistance methyltransferase from Micromonospora purpurea, producer of gentamicin complex. Two vectors were created that express protein with an N-terminal 6x histidine tag with and without an enterokinase recognition producing proteins His(6)-EK-GrmA and His(6)-GrmA, respectively. The activity of both recombinant proteins was demonstrated in vivo. After optimized expression and native purification both protein variants proved to be active in in vitro methylation assays. This work lays a foundation for future detailed biochemical, structural and pharmacological studies with this member of an important group of aminoglycoside-resistance enzymes.
PB  - Springer, New York
T2  - Protein Journal
T1  - Heterologous Escherichia coli Expression, Purification and Characterization of the GrmA Aminoglycoside-Resistance Methyltransferase
EP  - 332
IS  - 7-8
SP  - 326
VL  - 28
DO  - 10.1007/s10930-009-9197-9
ER  - 

@article{
author = "Morić, Ivana and Jeremić, Sanja and Savić, Miloje and Ilić-Tomić, Tatjana and Conn, Graeme L. and Vasiljević, Branka",
year = "2009",
abstract = "The mechanism of resistance to aminoglycosides based on methylation of their target, 16S rRNA, was until recently described only in antibiotic producing microorganisms. However, equivalent methyltransferases have now also been identified among numerous clinical Gram-negative pathogenic isolates. We have cloned, expressed, and purified GrmA, the aminoglycoside-resistance methyltransferase from Micromonospora purpurea, producer of gentamicin complex. Two vectors were created that express protein with an N-terminal 6x histidine tag with and without an enterokinase recognition producing proteins His(6)-EK-GrmA and His(6)-GrmA, respectively. The activity of both recombinant proteins was demonstrated in vivo. After optimized expression and native purification both protein variants proved to be active in in vitro methylation assays. This work lays a foundation for future detailed biochemical, structural and pharmacological studies with this member of an important group of aminoglycoside-resistance enzymes.",
publisher = "Springer, New York",
journal = "Protein Journal",
title = "Heterologous Escherichia coli Expression, Purification and Characterization of the GrmA Aminoglycoside-Resistance Methyltransferase",
pages = "332-326",
number = "7-8",
volume = "28",
doi = "10.1007/s10930-009-9197-9"
}

Morić, I., Jeremić, S., Savić, M., Ilić-Tomić, T., Conn, G. L.,& Vasiljević, B.. (2009). Heterologous Escherichia coli Expression, Purification and Characterization of the GrmA Aminoglycoside-Resistance Methyltransferase. in Protein Journal
Springer, New York., 28(7-8), 326-332.
https://doi.org/10.1007/s10930-009-9197-9

Morić I, Jeremić S, Savić M, Ilić-Tomić T, Conn GL, Vasiljević B. Heterologous Escherichia coli Expression, Purification and Characterization of the GrmA Aminoglycoside-Resistance Methyltransferase. in Protein Journal. 2009;28(7-8):326-332.
doi:10.1007/s10930-009-9197-9 .

Morić, Ivana, Jeremić, Sanja, Savić, Miloje, Ilić-Tomić, Tatjana, Conn, Graeme L., Vasiljević, Branka, "Heterologous Escherichia coli Expression, Purification and Characterization of the GrmA Aminoglycoside-Resistance Methyltransferase" in Protein Journal, 28, no. 7-8 (2009):326-332,
https://doi.org/10.1007/s10930-009-9197-9 . .

TY  - JOUR
AU  - Savić, Miloje
AU  - Lovrić, Josip
AU  - Ilić-Tomić, Tatjana
AU  - Vasiljević, Branka
AU  - Conn, Graeme L.
PY  - 2009
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/375
AB  - 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.
PB  - Oxford Univ Press, Oxford
T2  - Nucleic Acids Research
T1  - Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics
EP  - 5431
IS  - 16
SP  - 5420
VL  - 37
DO  - 10.1093/nar/gkp575
ER  - 

@article{
author = "Savić, Miloje and Lovrić, Josip and Ilić-Tomić, Tatjana and Vasiljević, Branka and Conn, Graeme L.",
year = "2009",
abstract = "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.",
publisher = "Oxford Univ Press, Oxford",
journal = "Nucleic Acids Research",
title = "Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics",
pages = "5431-5420",
number = "16",
volume = "37",
doi = "10.1093/nar/gkp575"
}

Savić, M., Lovrić, J., Ilić-Tomić, T., Vasiljević, 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. in Nucleic Acids Research
Oxford Univ Press, Oxford., 37(16), 5420-5431.
https://doi.org/10.1093/nar/gkp575

Savić M, Lovrić J, Ilić-Tomić T, Vasiljević B, Conn GL. Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics. in Nucleic Acids Research. 2009;37(16):5420-5431.
doi:10.1093/nar/gkp575 .

Savić, Miloje, Lovrić, Josip, Ilić-Tomić, Tatjana, Vasiljević, Branka, Conn, Graeme L., "Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics" in Nucleic Acids Research, 37, no. 16 (2009):5420-5431,
https://doi.org/10.1093/nar/gkp575 . .

TY  - 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 . .

TY  - JOUR
AU  - Ilić-Tomić, Tatjana
AU  - Morić, Ivana
AU  - Conn, Graeme L.
AU  - Vasiljević, Branka
PY  - 2008
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/324
AB  - The aminoglycoside resistance genes sgm from Micromonospora zionensis and kgmB from Streptomyces tenebrarius were cloned into a yeast expression vector to test whether the encoded prokaryotic methylases can modify the 18S rRNA A-site and thus confer resistance to G-418. Despite the detectable presence of mRNAs in yeast cells, neither G-418-resistant yeast transformants nor positive western blot signals were obtained. Neither methylase was capable of methylating 40S subunits despite very high conservation of the antibiotic rRNA binding sites. However, the results provide novel insight into the action of Sgm by showing that it methylates the same site as KgmB, i.e. G1405 in 16S rRNA.
PB  - Elsevier Science Bv, Amsterdam
T2  - Research in Microbiology
T1  - Aminoglycoside resistance genes sgm and kgmB protect bacterial but not yeast small ribosomal subunits in vitro despite high conservation of the rRNA A-site
EP  - 662
IS  - 9-10
SP  - 658
VL  - 159
DO  - 10.1016/j.resmic.2008.09.006
ER  - 

@article{
author = "Ilić-Tomić, Tatjana and Morić, Ivana and Conn, Graeme L. and Vasiljević, Branka",
year = "2008",
abstract = "The aminoglycoside resistance genes sgm from Micromonospora zionensis and kgmB from Streptomyces tenebrarius were cloned into a yeast expression vector to test whether the encoded prokaryotic methylases can modify the 18S rRNA A-site and thus confer resistance to G-418. Despite the detectable presence of mRNAs in yeast cells, neither G-418-resistant yeast transformants nor positive western blot signals were obtained. Neither methylase was capable of methylating 40S subunits despite very high conservation of the antibiotic rRNA binding sites. However, the results provide novel insight into the action of Sgm by showing that it methylates the same site as KgmB, i.e. G1405 in 16S rRNA.",
publisher = "Elsevier Science Bv, Amsterdam",
journal = "Research in Microbiology",
title = "Aminoglycoside resistance genes sgm and kgmB protect bacterial but not yeast small ribosomal subunits in vitro despite high conservation of the rRNA A-site",
pages = "662-658",
number = "9-10",
volume = "159",
doi = "10.1016/j.resmic.2008.09.006"
}

Ilić-Tomić, T., Morić, I., Conn, G. L.,& Vasiljević, B.. (2008). Aminoglycoside resistance genes sgm and kgmB protect bacterial but not yeast small ribosomal subunits in vitro despite high conservation of the rRNA A-site. in Research in Microbiology
Elsevier Science Bv, Amsterdam., 159(9-10), 658-662.
https://doi.org/10.1016/j.resmic.2008.09.006

Ilić-Tomić T, Morić I, Conn GL, Vasiljević B. Aminoglycoside resistance genes sgm and kgmB protect bacterial but not yeast small ribosomal subunits in vitro despite high conservation of the rRNA A-site. in Research in Microbiology. 2008;159(9-10):658-662.
doi:10.1016/j.resmic.2008.09.006 .

Ilić-Tomić, Tatjana, Morić, Ivana, Conn, Graeme L., Vasiljević, Branka, "Aminoglycoside resistance genes sgm and kgmB protect bacterial but not yeast small ribosomal subunits in vitro despite high conservation of the rRNA A-site" in Research in Microbiology, 159, no. 9-10 (2008):658-662,
https://doi.org/10.1016/j.resmic.2008.09.006 . .