Information on EC 2.1.1.182 - 16S rRNA (adenine1518-N6/adenine1519-N6)-dimethyltransferase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.1.1.182
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RECOMMENDED NAME
GeneOntology No.
16S rRNA (adenine1518-N6/adenine1519-N6)-dimethyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
4 S-adenosyl-L-methionine + adenine1518/adenine1519 in 16S rRNA = 4 S-adenosyl-L-homocysteine + N6-dimethyladenine1518/N6-dimethyladenine1519 in 16S rRNA
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:16S rRNA (adenine1518-N6/adenine1519-N6)-dimethyltransferase
KsgA introduces the most highly conserved ribosomal RNA modification, the dimethylation of adenine1518 and adenine1519 in 16S rRNA. Strains lacking the methylase are resistant to kasugamycin [1].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SwissProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
strain 6BC
UniProt
Manually annotated by BRENDA team
strain 6BC
UniProt
Manually annotated by BRENDA team
strain L2
UniProt
Manually annotated by BRENDA team
strain L2
UniProt
Manually annotated by BRENDA team
strain HB8
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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the KsgA family belongs tothe group of S-adenosyl-L-methionine-dependent methyltransferases, known as class I MTases, KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. Part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of S-adenosyl-L-methionine-dependent methyltransferases
malfunction
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4 S-adenosyl-L-methionine + adenine1518/1519 in 18S rRNA
4 S-adenosyl-L-homocysteine + N6-dimethyladenine1518/N6-dimethyladenine1519 in 18S rRNA
show the reaction diagram
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methylation of Escherichia coli 30S ribosomes. Under assay conditions the enzyme produces both N6-methyladenine and N6-dimethyladenine, with 0.8times as much N6-methyladenine as N6-dimethyladenine
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-
?
4 S-adenosyl-L-methionine + adenine1518/adenine1519 in 16S rRNA
4 S-adenosyl-L-homocysteine + N6-dimethyladenine1518/N6-dimethyladenine1519 in 16S rRNA
show the reaction diagram
S-adenosyl-L-methionine + adenine1518/adenine1519 in 16S rRNA
S-adenosyl-L-homocysteine + N6-dimethyladenine1518/N6-dimethyladenine1519 in 16S rRNA
show the reaction diagram
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
4 S-adenosyl-L-methionine + adenine1518/adenine1519 in 16S rRNA
4 S-adenosyl-L-homocysteine + N6-dimethyladenine1518/N6-dimethyladenine1519 in 16S rRNA
show the reaction diagram
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-
-
-
?
S-adenosyl-L-methionine + adenine1518/adenine1519 in 16S rRNA
S-adenosyl-L-homocysteine + N6-dimethyladenine1518/N6-dimethyladenine1519 in 16S rRNA
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl-L-methionine
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-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KCl
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slightly stimulated up to 0.2 M
NaCl
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slightly stimulated up to 0.2 M
additional information
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K+ and NH4+ in the range of 10 to 150 mM have little effect on methylation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
protein S21
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S21 probably inhibits KsgA activity in an indirect way, presumably by stabilizing 30S in a conformation that for whatever reason cannot be methylated by KsgA
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additional information
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a mixture of 30 S ribosomal proteins inhibits methylation of mutant 30 S ribosomes. This inhibition can be ascribed to ribosomal protein S21. Initiation factor 3 partially inhibits methylation of mutant 30 S ribosomes
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
-
KM-value for S-adenosylmethionine is in the range 0.002-0.007 mM
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
Escherichia coli
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IC50 values of unlabeled wild-type or mutant protein competing with fluorescently labeled wild-type protein for binding to 30S subunits
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.1 - 7.2
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at 34°C
7.4
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8 - 7.8
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change of pH from 7.8 to 6.8 reduces methylation by 50%
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
37
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assay at
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Burkholderia pseudomallei (strain K96243)
Coxiella burnetii (strain RSA 493 / Nine Mile phase I)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Rickettsia bellii (strain RML369-C)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30385
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x * 30385, calculated from sequence
30400
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x * 30400, SDS-PAGE, calculated from sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 30000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
structures of KsgA, in its ligand-free form, in complex with RNA and in complex with both RNA and S-adenosylhomocysteine
the structure is solved to a resolution of 2.1 A
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microbatch technique under oil at 4°C, crystal structures of KsgA in the apo form and with 5’-methylthioadenosine or adenosine bound in the cofactor-binding site
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant wild-type and mutant enzymes from Escherichia coli strain XL1-Blue
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of ksgA from the 431DELTA99 KSGR mutant
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expression of a pGEX-KsgA fusion construct in Escherichia coli KSR7
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expression of the Chlamydia trachomatis L2 KsgA ortholog restored kasugamycin sensitivity to the Escherichia coli ksgA mutant
expression of wild-type and mutant enzymes in Escherichia coli strain XL1-Blue
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full-length ksgA gene is cloned into the expression vector pET26b and overexpressed in Escherichia coli strain BL21Star
KsgA engineered with an N-terminal polyhistidine tag is overexpressed in Escherichia coli cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C168A
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site-directed mutagenesis, the mutation does not affect KsgA activity
E43A
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mutation located in the S-adenosylmethionine-binding motifs severely reduces methyltransferase activity, the mutation retains the ability to suppress the growth defect of the Era(E200K) strain at a low temperature
G47A
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mutation located in the S-adenosylmethionine-binding motifs severely reduces methyltransferase activity, the mutation retains the ability to suppress the growth defect of the Era(E200K) strain at a low temperature
L114P
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site-directed mutagenesis of the active site residue, the KsgA mutant shows diminished overall activity, and impaired ability to methylate the N6-methyladenosine intermediate to produce N6,N6-dimethyladenosine. Reduced activity is not due to disruption of 30S substrate binding
N113A
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site-directed mutagenesis of the active site residue, the KsgA mutant shows diminishes activity to a level comparable to L114P without affecting the methylation of N6-methyladenosine. Reduced activity is not due to disruption of 30S substrate binding
R248A
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mutation at the C-terminal does not affect the methyltransferase activity and fails to suppress the growth defect of the Era(E200K) strain
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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