Literature summary for 2.1.1.184 extracted from

Maravic, G.; Bujnicki, J.M.; Feder, M.; Pongor, S.; Fl�gel, M.
Alanine-scanning mutagenesis of the predicted rRNA-binding domain of ErmC' redefines the substrate-binding site and suggests a model for protein-RNA interactions (2003), Nucleic Acids Res., 31, 4941-4949.

Search for more literature for 2.1.1.184

Application

Application	Comment	Organism
medicine	the Erm family of adenine-N6 methyltransferases is responsible for the development of resistance to macrolide-lincosamide-streptogramin B antibiotics through the methylation of 23S ribosomal RNA. Hence, these proteins are important potential drug targets	Bacillus subtilis

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Bacillus subtilis

Protein Variants

Protein Variants	Comment	Organism
E202A	mutant of a residues positioned on the surface of the small domain, does not display substantial defects in activity compared with the wild-type enzyme. kcat/Km for S-adenosyl-L-methionine is 32% of the wild-type value. kcat/Km for RNA is 40% of wild-type value	Bacillus subtilis
K133A	decreased ability in rendering DH5alpha cells resistant to erythromycin, suggesting that this amino acid is not absolutely indispensable for the ErmC' activity, but might be involved in important RNA-protein interactions. No change in affinity towards the RNA substrate. kcat/Km for S-adenosyl-L-methionine is% of the wild-type value. kcat/Km for RNA is 27% of wild-type value. kcat/Km for S-adenosyl-L-methionine is 13% of the wild-type value. kcat/Km for RNA is% of wild-type value	Bacillus subtilis
K197A	mutant of a residues positioned on the surface of the small domain, does not display substantial defects in activity compared with the wild-type enzyme. kcat/Km for S-adenosyl-L-methionine is 67% of the wild-type value. kcat/Km for RNA is 50% of wild-type value	Bacillus subtilis
K197A/N200A/E202A/K204A/K205A	the five mutations together do not show a visible cumulative effect on the ErmC' activity in vivo	Bacillus subtilis
K204A	mutant of a residues positioned on the surface of the small domain, does not display substantial defects in activity compared with the wild-type enzyme. kcat/Km for S-adenosyl-L-methionine is 73% of the wild-type value. kcat/Km for RNA is 89% of wild-type value	Bacillus subtilis
K205A	mutant of a residues positioned on the surface of the small domain, does not display substantial defects in activity compared with the wild-type enzyme. kcat/Km for S-adenosyl-L-methionine is 63% of the wild-type value. kcat/Km for RNA is 92% of wild-type value	Bacillus subtilis
K209A	mutant of a residues positioned on the surface of the small domain, does not display substantial defects in activity compared with the wild-type enzyme. kcat/Km for S-adenosyl-L-methionine is 72% of the wild-type value. kcat/Km for RNA is 73% of wild-type value	Bacillus subtilis
M196A	mutant of a residues positioned on the surface of the small domain, does not display substantial defects in activity compared with the wild-type enzyme. kcat/Km for S-adenosyl-L-methionine is 42% of the wild-type value. kcat/Km for RNA is 56% of wild-type value	Bacillus subtilis
additional information	With the aim of identification of essential protein-RNA interactions, charged side chains on the predicted target-binding surface of ErmC' are replaced systematically with alanine and the function of the single- and multiple-site mutants is studied in vitro and in vivo. kcat/Km for S-adenosyl-L-methionine is 21% of the wild-type value. kcat/Km for RNA is 17% of wild-type value	Bacillus subtilis
N192A	mutant of a residues positioned on the surface of the small domain, does not display substantial defects in activity compared with the wild-type enzyme. kcat/Km for S-adenosyl-L-methionine is 73% of the wild-type value. kcat/Km for RNA is 74% of wild-type value	Bacillus subtilis
N200A	mutant of a residues positioned on the surface of the small domain, does not display substantial defects in activity compared with the wild-type enzyme. kcat/Km for S-adenosyl-L-methionine is 42% of the wild-type value. kcat/Km for RNA is 54% of wild-type value	Bacillus subtilis
R112A	decreased ability in rendering DH5alpha cells resistant to erythromycin, suggesting that this amino acid is not absolutely indispensable for the ErmC' activity, but might be involved in important RNA-protein interactions. kcat/Km for S-adenosyl-L-methionine is 12% of the wild-type value. kcat/Km for RNA is 7% of wild-type value	Bacillus subtilis
R112D	decreased ability in rendering DH5alpha cells resistant to erythromycin. The R112D mutant shows a more pronounced decrease in RNA-binding affinity compared with R112A. kcat/Km for S-adenosyl-L-methionine is 5% of the wild-type value. kcat/Km for RNA is 2% of wild-type value	Bacillus subtilis
R134A	mutant exhibits the most severe effect on the ErmC' ability to generate erythromycin resistance, this mutant has completely lost the activity in vivo. kcat/Km for S-adenosyl-L-methionine is 3% of the wild-type value. kcat/Km for RNA is 1% of wild-type value	Bacillus subtilis
R140A	decreased ability in rendering DH5alpha cells resistant to erythromycin, suggesting that this amino acid is not absolutely indispensable for the ErmC' activity, but might be involved in important RNA-protein interactions. kcat/Km for S-adenosyl-L-methionine is 1% of the wild-type value. kcat/Km for RNA is 3% of wild-type value	Bacillus subtilis
T108A	decreased ability in rendering DH5alpha cells resistant to erythromycin, suggesting that this amino acid is not absolutely indispensable for the ErmC' activity, but might be involved in important RNA-protein interactions. kcat/Km for S-adenosyl-L-methionine is 4% of the wild-type value. kcat/Km for RNA is 3% of wild-type value	Bacillus subtilis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.00043	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme T108A	Bacillus subtilis
0.00057	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme E202A	Bacillus subtilis
0.00062	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme M196A	Bacillus subtilis
0.00069	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, wild-type enzyme	Bacillus subtilis
0.00079	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme N192A	Bacillus subtilis
0.00089	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K204A	Bacillus subtilis
0.00089	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme N200A	Bacillus subtilis
0.00091	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K205A	Bacillus subtilis
0.00095	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K209A	Bacillus subtilis
0.00121	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K197A/N200A/E202A/K204A/K205A	Bacillus subtilis
0.00141	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme R140A	Bacillus subtilis
0.00159	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K197A	Bacillus subtilis
0.0017	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme R112A	Bacillus subtilis
0.00186	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K133A	Bacillus subtilis
0.002	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme N192A	Bacillus subtilis
0.0025	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme R134A	Bacillus subtilis
0.0028	-	S-adenosyl-L-methionine	pH 7.5, 25°C, wild-type enzyme	Bacillus subtilis
0.003	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K197A	Bacillus subtilis
0.003	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme M196A	Bacillus subtilis
0.00364	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme R112D	Bacillus subtilis
0.0038	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K204A	Bacillus subtilis
0.0044	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K205A	Bacillus subtilis
0.0046	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K209A	Bacillus subtilis
0.0058	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme R112A	Bacillus subtilis
0.006	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K133A	Bacillus subtilis
0.0066	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K197A/N200A/E202A/K204A/K205A	Bacillus subtilis
0.007	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme N200A	Bacillus subtilis
0.0098	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme E202A	Bacillus subtilis
0.011	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme R112D	Bacillus subtilis
0.021	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme R134A	Bacillus subtilis
0.022	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme R140A	Bacillus subtilis
0.0257	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme T108A	Bacillus subtilis

Organism

Organism	UniProt	Comment	Textmining
Bacillus subtilis	P13956	carrying naturally occurring plasmid pIM13 with ermC' gene	-
Bacillus subtilis BD1167	P13956	carrying naturally occurring plasmid pIM13 with ermC' gene	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant enzyme	Bacillus subtilis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
2 S-adenosyl-L-methionine + adenine2085 in 23S rRNA	-	Bacillus subtilis	2 S-adenosyl-L-homocysteine + N6-dimethyladenine2085 in 23S rRNA	-	?
2 S-adenosyl-L-methionine + adenine2085 in 23S rRNA	-	Bacillus subtilis BD1167	2 S-adenosyl-L-homocysteine + N6-dimethyladenine2085 in 23S rRNA	-	?

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Bacillus subtilis

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.00011	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme T108A	Bacillus subtilis
0.00021	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme R134A	Bacillus subtilis
0.00034	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme R140A	Bacillus subtilis
0.00049	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme R112D	Bacillus subtilis
0.00113	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme R112A	Bacillus subtilis
0.00193	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K197A/N200A/E202A/K204A/K205A	Bacillus subtilis
0.00211	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme E202A	Bacillus subtilis
0.00222	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K133A	Bacillus subtilis
0.00318	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme M196A	Bacillus subtilis
0.00439	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme N200A	Bacillus subtilis
0.00544	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme N192A	Bacillus subtilis
0.00637	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, wild-type enzyme	Bacillus subtilis
0.00637	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K209A	Bacillus subtilis
0.00729	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K204A	Bacillus subtilis
0.00731	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K197A	Bacillus subtilis
0.00766	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K205A	Bacillus subtilis
0.0121	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme R112D	Bacillus subtilis
0.0149	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme R140A	Bacillus subtilis
0.0157	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme R134A	Bacillus subtilis
0.0161	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme R112A	Bacillus subtilis
0.0229	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme T108A	Bacillus subtilis
0.0314	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K197A/N200A/E202A/K204A/K205A	Bacillus subtilis
0.0318	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme M196A	Bacillus subtilis
0.0341	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme N192A	Bacillus subtilis
0.038	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K133A	Bacillus subtilis
0.0522	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K197A	Bacillus subtilis
0.0648	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K204A	Bacillus subtilis
0.0649	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K205A	Bacillus subtilis
0.0655	-	S-adenosyl-L-methionine	pH 7.5, 25°C, wild-type enzyme	Bacillus subtilis
0.0688	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme N200A	Bacillus subtilis
0.0735	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme E202A	Bacillus subtilis
0.0775	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K209A	Bacillus subtilis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Bacillus subtilis

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.066	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme R112A	Bacillus subtilis
0.084	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme R134A	Bacillus subtilis
0.135	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme R112D	Bacillus subtilis
0.241	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme R140A	Bacillus subtilis
0.258	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme T108A	Bacillus subtilis
0.67	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme R140A	Bacillus subtilis
0.74	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme R134A	Bacillus subtilis
0.89	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme T108A	Bacillus subtilis
1.12	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme R112D	Bacillus subtilis
1.193	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K133A	Bacillus subtilis
1.47	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K205A	Bacillus subtilis
1.6	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K197A/N200A/E202A/K204A/K205A	Bacillus subtilis
2.78	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme R112A	Bacillus subtilis
3.7	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme E202A	Bacillus subtilis
4.6	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K197A	Bacillus subtilis
4.79	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K197A/N200A/E202A/K204A/K205A	Bacillus subtilis
4.9	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme N200A	Bacillus subtilis
5.13	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme M196A	Bacillus subtilis
6.33	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K133A	Bacillus subtilis
6.71	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K209A	Bacillus subtilis
6.89	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme N192A	Bacillus subtilis
7.53	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme E202A	Bacillus subtilis
8.19	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K204A	Bacillus subtilis
8.42	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, mutant enzyme K205A	Bacillus subtilis
9.23	-	adenine2085 in 23S rRNA	pH 7.5, 25°C, wild-type enzyme	Bacillus subtilis
9.64	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme M196A	Bacillus subtilis
9.7	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme N200A	Bacillus subtilis
15.63	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K197A	Bacillus subtilis
16.77	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K209A	Bacillus subtilis
16.92	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme K204A	Bacillus subtilis
17.05	-	S-adenosyl-L-methionine	pH 7.5, 25°C, mutant enzyme N192A	Bacillus subtilis
23.23	-	S-adenosyl-L-methionine	pH 7.5, 25°C, wild-type enzyme	Bacillus subtilis