Information on EC 2.3.1.82 - aminoglycoside 6'-N-acetyltransferase

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The expected taxonomic range for this enzyme is: Bacteria

EC NUMBER
COMMENTARY hide
2.3.1.82
-
RECOMMENDED NAME
GeneOntology No.
aminoglycoside 6'-N-acetyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
acetyl-CoA + kanamycin-B = CoA + N6'-acetylkanamycin-B
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acyl group transfer
SYSTEMATIC NAME
IUBMB Comments
acetyl-CoA:kanamycin-B N6'-acetyltransferase
The antibiotics kanamycin A, kanamycin B, neomycin, gentamicin C1a, gentamicin C2 and sisomicin are substrates. The antibiotic tobramycin, but not paromomycin, can also act as acceptor. The 6-amino group of the purpurosamine ring is acetylated.
CAS REGISTRY NUMBER
COMMENTARY hide
56467-65-3
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain 8
-
-
Manually annotated by BRENDA team
strain 8
-
-
Manually annotated by BRENDA team
strain CFr564, enzyme variant with Ser119
-
-
Manually annotated by BRENDA team
strain CFr564, enzyme variant with Ser119
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
strain EC1562 and EC1563, enzyme variant with Ser119
-
-
Manually annotated by BRENDA team
strain EC1562 and EC1563, enzyme variant with Ser119
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
CS2R2
-
-
Manually annotated by BRENDA team
NR79
-
-
Manually annotated by BRENDA team
strains 2513 and 731
-
-
Manually annotated by BRENDA team
strain BM2687, enzyme Ib
-
-
Manually annotated by BRENDA team
strain BM2687, enzyme Ib
-
-
Manually annotated by BRENDA team
strain VU12944/77
-
-
Manually annotated by BRENDA team
RYC 13036
-
-
Manually annotated by BRENDA team
RYC 13036
-
-
Manually annotated by BRENDA team
IFO14147
SwissProt
Manually annotated by BRENDA team
IFO14147
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetyl-CoA + 2'-N-ethylnetilmicin
CoA + N6'-acetyl-2'-N-ethylnetilmicin
show the reaction diagram
acetyl-CoA + amikacin
?
show the reaction diagram
-
-
-
-
?
acetyl-CoA + amikacin
CoA + 6'-N-acetylamikacin
show the reaction diagram
-
aminoglycoside acetyltransferase eis acetylates and inactivates amikacin
-
-
?
acetyl-CoA + amikacin
CoA + N6'-acetylamikacin
show the reaction diagram
acetyl-CoA + aminoglycoside
CoA + 6'-N-acetylaminoglycoside
show the reaction diagram
-
-
-
-
?
acetyl-CoA + butirosin
CoA + N6'-acetylbutirosin
show the reaction diagram
-
-
-
-
?
acetyl-CoA + ciprofloxacin
CoA + N4'-acetylnorfloxacin
show the reaction diagram
-
-
-
-
?
acetyl-CoA + dibekacin
CoA + N6'-acetyldibekacin
show the reaction diagram
acetyl-CoA + fortimicin A
CoA + N6'-acetylfortimicin A
show the reaction diagram
-
-
-
-
?
acetyl-CoA + gentamicin
CoA + 6'-N-acetylgentamicin
show the reaction diagram
-
-
-
?
acetyl-CoA + gentamicin
CoA + N6'-acetylgentamicin
show the reaction diagram
acetyl-CoA + gentamicin B
CoA + N6'-acetylgentamicin B
show the reaction diagram
-
-
-
-
?
acetyl-CoA + gentamicin C
CoA + N6'-acetylgentamicin C
show the reaction diagram
acetyl-CoA + gentamicin C1
CoA + N6'-acetylgentamicin C1
show the reaction diagram
-
the AAC(6')-Ib protein is unable to efficiently modify gentamicin C1, 1.7% relative activity to sisomicin, however it is capable of modifying amikacin, 65.5% relative activity to sisomycin. The mutant enzyme AAC(6')-Ib L119S shows a 2.8fold increase in acetylation of gentamicin C1, but a 8.7fold reduction in the ability to modify amikacin. The AAC(6')-IIa protein modifies gentamicin C1 at 10.1% relative activity to sisomicin, however it shows low activity towards amikacin, 4.1% activity relative to sisomycin. The mutation AAC(6')-IIa S119L results in a 4.8fold reduction in the acetylation of gentamicin C1, but causes an 2fold increase in the ability to modify amikacin
-
-
?
acetyl-CoA + gentamicin C1a
CoA + N6'-acetylgentamicin C1a
show the reaction diagram
acetyl-CoA + gentamicin C2
CoA + N6'-acetylgentgamicin C2
show the reaction diagram
-
poor substrate, not gentamicin A and C1
-
-
?
acetyl-CoA + histone
CoA + ?
show the reaction diagram
a mixture of calf histones enriched in H3 and H4
-
-
?
acetyl-CoA + human histone H3 peptide containing a C-terminal cysteine residue
CoA + ?
show the reaction diagram
-
-
-
-
?
acetyl-CoA + hybrimycin A1
CoA + N6'-acetylhybrimycin A1
show the reaction diagram
-
-
-
-
?
acetyl-CoA + hybrimycin A2
CoA + N6'-acetylhybrimycin A2
show the reaction diagram
-
-
-
-
?
acetyl-CoA + hybrimycin A3
CoA + N6'-acetylhybrimycin A3
show the reaction diagram
-
-
-
-
?
acetyl-CoA + hybrimycin B1
CoA + N6'-acetylhybrimycin B1
show the reaction diagram
-
-
-
-
?
acetyl-CoA + hybrimycin B2
CoA + N6'-acetylhybrimycin B2
show the reaction diagram
-
-
-
-
?
acetyl-CoA + hybrimycin B3
CoA + N6'-acetylhybrimycin B3
show the reaction diagram
-
-
-
-
?
acetyl-CoA + isepamicin
CoA + N6'-acetylisepamicin
show the reaction diagram
-
-
-
-
?
acetyl-CoA + istamycin-B
CoA + N6'-acetylistamycin-B
show the reaction diagram
-
-
-
-
?
acetyl-CoA + kanamycin
CoA + 6'-N-acetylkanamycin
show the reaction diagram
-
aminoglycoside acetyltransferase eis acetylates and inactivates kanamycin
-
-
?
acetyl-CoA + kanamycin
CoA + N6'-acetylkanamycin
show the reaction diagram
acetyl-CoA + kanamycin A
CoA + N6'-acetylkanamycin A
show the reaction diagram
acetyl-CoA + kanamycin B
CoA + N6'-acetylkanamycin B
show the reaction diagram
acetyl-CoA + lividomycin A
CoA + O6'-acetyllividomycin A
show the reaction diagram
-
-
-
?
acetyl-CoA + micromomicin
CoA + N6'-acetylmicromomicin
show the reaction diagram
acetyl-CoA + myelin basic protein
CoA + ?
show the reaction diagram
-
-
-
?
acetyl-CoA + neamine
CoA + 6'-N-acetylneamine
show the reaction diagram
acetyl-CoA + neamine
CoA + N6'-acetylneamine
show the reaction diagram
acetyl-CoA + nebramycin factor 4
CoA + N6'-acetylnebramycin factor 4
show the reaction diagram
-
no activity with factor 2
-
-
?
acetyl-CoA + nebramycin factor 6
CoA + N6'-acetylnebramycin factor 6
show the reaction diagram
-
no activity with factor 2
-
-
?
acetyl-CoA + neomycin
CoA + N6'-acetylneomycin
show the reaction diagram
acetyl-CoA + neomycin A
CoA + N6'-acetylneomycin A
show the reaction diagram
-
-
-
-
?
acetyl-CoA + neomycin B
CoA + ?
show the reaction diagram
-
-
-
?
acetyl-CoA + neomycin B
CoA + N6'-acetylneomycin B
show the reaction diagram
acetyl-CoA + neomycin C
CoA + N6'-acetylneomycin
show the reaction diagram
-
-
-
-
?
acetyl-CoA + neomycin C
CoA + N6'-acetylneomycin C
show the reaction diagram
acetyl-CoA + netilmicin
CoA + N6'-acetylnetilmicin
show the reaction diagram
acetyl-CoA + norfloxacin
CoA + N4'-acetylnorfloxacin
show the reaction diagram
-
-
-
-
?
acetyl-CoA + paromomycin
CoA + O6'-acetylparomomycin
show the reaction diagram
-
-
-
-
?
acetyl-CoA + poly-L-Lys
CoA + ?
show the reaction diagram
-
-
-
?
acetyl-CoA + ribostamycin
CoA + N6'-acetylribostamycin
show the reaction diagram
acetyl-CoA + ribostamycin
CoA + N6'acetylribostamycin
show the reaction diagram
-
-
-
-
?
acetyl-CoA + sisomicin
CoA + N6'-acetylsisomicin
show the reaction diagram
acetyl-CoA + tobramycin
CoA + N6'-acetyltobramycin
show the reaction diagram
butyryl-CoA + tobramycin
CoA + N6'-butyryltobramycin
show the reaction diagram
-
-
-
-
?
malonyl-CoA + sisomycin
CoA + ?
show the reaction diagram
-
-
-
?
malonyl-CoA + tobramycin
CoA + N6'-malonyltobramycin
show the reaction diagram
-
-
-
-
?
n-butyryl-CoA + sisomycin
CoA + ?
show the reaction diagram
-
-
-
?
n-propionyl-CoA + sisomycin
CoA + ?
show the reaction diagram
-
-
-
?
propionyl-CoA + tobramycin
CoA + N6'-propionyltobramycin
show the reaction diagram
-
tobramycin exhibits a rapid, tobramycin-independent rate of hydrolysis that is linearly proportional to enzyme
-
-
?
additional information
?
-
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
acetyl-CoA + aminoglycoside
CoA + 6'-N-acetylaminoglycoside
show the reaction diagram
-
-
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acetyl-CoA
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
-
required
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[(3-sulfanylpropanoyl)amino]-alpha-D-glucopyranoside-S-CoA
-
-
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[(4-sulfanylbutanoyl)amino]-alpha-D-glucopyranoside-S-CoA
-
-
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[(5-sulfanylpentanoyl)amino]-alpha-D-glucopyranoside-S-CoA
-
-
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[(sulfanylacetyl)amino]-alpha-D-glucopyranoside-S-CoA
-
-
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[[(2-sulfanylethyl)sulfonyl]amino]-alpha-D-glucopyranoside-S-CoA
-
-
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[[(dioxidosulfanyl)acetyl]amino]-alpha-D-glucopyranoside-S-CoA
-
-
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[[(oxidosulfanyl)acetyl]amino]-alpha-D-glucopyranoside-S-CoA
-
-
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[[3-(dioxidosulfanyl)propanoyl]amino]-alpha-D-glucopyranoside-S-CoA
-
-
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[[3-(oxidosulfanyl)propanoyl]amino]-alpha-D-glucopyranoside-S-CoA
-
-
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2-deoxy-6-O-[methoxy(2-sulfanylethyl)phosphoryl]-alpha-D-glucopyranoside-S-CoA
-
-
1-(bromomethyl)phenanthrene
-
inactivates through covalent modification of Asp99
6'-N-acetylkanamycin A
-
competitive versus kanamycin A and noncompetitive/mixed versus acetyl-CoA
6'-N-acetylneamine
-
product inhibition
amikacin
-
substrate inhibition
Butyryl-CoA
CoA
-
product inhibition
CoA-aminoglycoside 1
-
-
CoA-aminoglycoside 11a
-
-
CoA-aminoglycoside 11b
-
-
CoA-aminoglycoside 11c
-
-
CoA-aminoglycoside 1a
-
-
CoA-aminoglycoside 1b
-
-
CoA-aminoglycoside 1c
-
-
CoA-aminoglycoside 1d
-
-
CoA-aminoglycoside 2
-
-
CoA-aminoglycoside 3
-
-
CoA-aminoglycoside 4a
-
competitive
CoA-aminoglycoside 4b
-
competitive
CoA-aminoglycoside 4c
-
competitive
CoA-aminoglycoside 4d
-
competitive
CoA-aminoglycoside 4e
-
competitive
CoA-aminoglycoside 4f
-
competitive
CoA-aminoglycoside 5b
-
competitive
CoA-aminoglycoside 5d
-
competitive
CoA-aminoglycoside 5e
-
competitive
dithio-CoA
-
-
gentamicin
-
substrate inhibition
gentamicin A
-
-
gentamicin C1
gentamine A
-
-
iodoacetamide
-
inactivation in a biphasic manner, half of the activity is lost rapidly and the other half more slowly, tobramycin but not acetyl-CoA protects
kanamycin A
-
-
kanamycin B
-
-
lividomycin
-
-
lividomycin A
-
-
N6'-acetyl kanamycin A
-
-
N6'-Acetylgentamicin C1a
-
-
N6'-acetylneomycin B
-
-
netilmicin
paromamine
-
-
paromomycin
Pefloxacin
-
-
poly-L-Lys
substrate inhibition
tobramycin
additional information
-
no inhibition by GTP
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0033 - 0.154
acetyl-CoA
0.001 - 2.19
amikacin
0.014
butirosin
-
pH 6.0, 37C
9.3
Butyryl-CoA
-
reaction with tobramycin, wild-type enzyme
0.07
Ciprofloxacin
-
AAC(6')-Ib-cr variant
0.00038 - 0.036
dibekacin
0.0254 - 0.07
fortimicin A
0.0083 - 0.653
gentamicin
0.0223
gentamicin B
-
pH 6.0, 37C
0.0082
gentamicin C
-
wild-type enzyme
0.0003
isepamicin
-
reaction with AAC (6?)-Ib' domain
0.008 - 0.154
kanamycin
0.001 - 3.49
kanamycin A
0.00027 - 0.9
kanamycin B
0.04188
lividomycin A
pH 8.0, 30C
0.58 - 0.82
malonyl-CoA
0.0686
n-Butyryl-CoA
pH 8.0, 30C, cosubstrate: sisomycin
0.0161
n-propionyl-CoA
pH 8.0, 30C, cosubstrate: sisomycin
0.0058 - 0.509
neamine
0.0012 - 0.0573
neomycin
0.53
neomycin A
-
-
0.00052 - 0.02
neomycin B
0.00531 - 0.25
neomycin C
0.001 - 2.44
netilmicin
0.067
Norfloxacin
-
AAC(6')-Ib-cr variant
0.324 - 0.771
paromomycin
0.038
poly-L-Lys
pH 7.5, 37C
0.0195 - 0.27
propionyl-CoA
0.00908 - 0.306
ribostamycin
0.00324 - 0.012
sisomicin
0.216
tobramicin
-
pH 5.5, 37C
0.00353 - 1.15
tobramycin
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01 - 5.23
acetyl-CoA
0.003 - 3.5
amikacin
0.466
butirosin
Enterococcus faecium
-
pH 6.0, 37C
1.03
Ciprofloxacin
Escherichia coli
-
AAC(6')-Ib-cr variant
0.632 - 1.3
dibekacin
0.0002 - 0.45
fortimicin A
0.5 - 2
gentamicin
0.388
gentamicin B
Enterococcus faecium
-
pH 6.0, 37C
1.01
gentamicin C
Enterococcus faecium
-
wild-type enzyme
2.4
isepamicin
Pseudomonas aeruginosa
-
reaction with AAC (6')-Ib' domain
0.01 - 2.4
kanamycin A
1.08 - 5.25
kanamycin B
0.002 - 1.3
neamine
0.001 - 6
neomycin
3.08
neomycin B
Escherichia coli
-
AAC(6')-Ib, wild type
0.135 - 0.205
neomycin C
0.2 - 1.3
netilmicin
1.08
Norfloxacin
Escherichia coli
-
AAC(6')-Ib-cr variant
0.0004 - 0.059
paromomycin
0.0019
poly-L-Lys
Enterococcus faecium
Q47764
pH 7.5, 37C
0.317
propionyl-CoA
Enterococcus faecium
-
pH 6.0, 37C
0.01 - 0.35
ribostamycin
0.37
sisomicin
Enterococcus faecium
-
pH 6.0, 37C
0.004 - 1.11
tobramycin
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00004
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[(3-sulfanylpropanoyl)amino]-alpha-D-glucopyranoside-S-CoA
-
-
0.00016
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[(4-sulfanylbutanoyl)amino]-alpha-D-glucopyranoside-S-CoA
-
-
0.008
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[(5-sulfanylpentanoyl)amino]-alpha-D-glucopyranoside-S-CoA
-
-
0.00008
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[(sulfanylacetyl)amino]-alpha-D-glucopyranoside-S-CoA
-
-
0.0016
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[[(2-sulfanylethyl)sulfonyl]amino]-alpha-D-glucopyranoside-S-CoA
0.00027
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[[(dioxidosulfanyl)acetyl]amino]-alpha-D-glucopyranoside-S-CoA
-
competitive inhibition
0.00006
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[[(oxidosulfanyl)acetyl]amino]-alpha-D-glucopyranoside-S-CoA
-
competitive inhibition
0.00009
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[[3-(dioxidosulfanyl)propanoyl]amino]-alpha-D-glucopyranoside-S-CoA
-
competitive inhibition
0.002
(1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2-amino-2,6-dideoxy-6-[[3-(oxidosulfanyl)propanoyl]amino]-alpha-D-glucopyranoside-S-CoA
0.04 - 0.55
6'-N-acetylated kanamycin A
0.15 - 0.385
6'-N-acetylkanamycin A
0.00499 - 0.02206
amikacin
6
AMP
-
pH 6.9, 37C, inhibition of the reaction with neamine
0.025 - 0.57
Butyryl-CoA
0.0036
CoA
-
pH 6.9, 37C
0.000076
CoA-aminoglycoside 1
-
-
0.000043
CoA-aminoglycoside 11a
-
-
0.000161
CoA-aminoglycoside 11b
-
-
0.00799
CoA-aminoglycoside 11c
-
-
0.000076
CoA-aminoglycoside 1a
-
-
0.000043
CoA-aminoglycoside 1b
-
-
0.000161
CoA-aminoglycoside 1c
-
-
0.008
CoA-aminoglycoside 1d
-
-
0.00011 - 0.000111
CoA-aminoglycoside 2
0.000119
CoA-aminoglycoside 3
-
-
0.0034
CoA-aminoglycoside 4a
-
-
0.13
CoA-aminoglycoside 4b
-
-
0.0012
CoA-aminoglycoside 4c
-
-
0.0034
CoA-aminoglycoside 4d
-
-
0.0036
CoA-aminoglycoside 4e
-
-
0.0074
CoA-aminoglycoside 4f
-
-
0.012
CoA-aminoglycoside 5b
-
-
0.0022
CoA-aminoglycoside 5d
-
-
0.011
CoA-aminoglycoside 5e
-
-
0.02 - 0.2
CoASH
0.072
dibekacin
-
pH 6.0, 37C
0.02256 - 0.079
gentamicin
4
gentamicin C1
-
pH 6.9, 37C, inhibition of the reaction with neamine
0.196
kanamycin A
-
pH 6.0, 37C
0.117
kanamycin B
-
pH 6.0, 37C
0.06446 - 0.168
netilmicin
0.0012 - 7.7
paromomycin
1.78
poly-L-Lys
pH 7.5, 37C
0.063
sisomicin
-
pH 6.0, 37C
0.00059 - 0.059
tobramycin
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
equilibrium binding and the directly determined thermodynamic parameters for different aminoglycosides and acyl-CoA derivatives to the wild-type enzyme and two mutant enzymes c109A and C109A/C70A using fluorescence spectroscopy and isothermal titration calorimetry
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.3
-
reaction with tobramycin, gentamicin C1a or neomycin
5.6
-
AAC(6')-Ib
5.8
-
reaction with kanamycin, neomycin, hybrimycin, nebramycin factor 4 and 6
8
-
activity assay
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.3 - 7.6
-
pH 5.3: about 50% of maximal activity, pH 7.6: about 20% of maximal activity, kanamycin A
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
-
activity assay at room temperature
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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evenly distributed
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
21000
determined by SDS-PAGE and Western Blot analysis
35000
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gel filtration
55000
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sucrose density gradient centrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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the enzyme catalyzes its own alpha-N-acetylation after posttranslational enzymaric deformylation and removal of the initiator Met
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystallized with its cofactor coenzyme A in space group C222(1), with unit cell parameters a = 71.5, b = 127.4, c = 76.9 A and one physiologically relevant dimer species per asymmetric unit
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hanging drop vapor diffusion technique, crystal structure of the 6'-N-acetyltransferase type li in complex with acetyl-CoA determined at 2.7 A resolution
the structure of AAC(6')-Ib is determined in various complexes with donor and acceptor ligands to resolutions greater than 2.2 A
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recombinant enzyme, vapor diffusion under oil, CoA-ribostamycin ternary complex
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vapor diffusion under oil, crystal structure of the AAC(6')-Iy-CoA-S-monomethyl-N-6'-acetylneamine complex
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small-angle X-ray scattering analysis shows that the enzyme adopts a rigid conformation in solution, where the N-terminal acetyltransferase domain is fixed to the C-terminal phosphotransferase domain and not loosely tethered. The addition of acetyl-coenzyme A, coenzyme A, GDP, guanosine 5'-[beta,gamma-imido]triphosphate, and combinations thereof to the protein result in only modest changes to the radius of gyration of the enzyme, which are not consistent with any large changes in enzyme structure upon binding. These results imply some selective advantage to the bifunctional enzyme beyond coexpression as a single polypeptide, likely linked to an improvement in enzymatic properties. The rigid structure may contribute to improved electrostatic steering of aminoglycoside substrates toward the two active sites, which may provide such an advantage
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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pH 7.4, 10 mM MgCl2, 0.6 mM 2-mercaptoethanol, about 35% loss of activity after 10 min, acetyl-CoA prevents, kanamycin protects slightly
42
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pH 7.4, 10 mM MgCl2, 0.6 mM 2-mercaptoethanol, about 70% loss of activity after 10 min, acetyl-CoA prevents, kanamycin protects slightly
additional information
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GTP, CoA, gentamicin C, neamine protect against thermal inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dialysis in the absence of Mg2+ causes irreversible loss of activity
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GTP, CoA, gentamicin C, neamine protect against thermal inactivation
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Mg2+ stabilizes
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, several months
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-30C, stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
both acetyltransferase domains of the bifunctional enzyme AAC(3)-Ib/AAC(6')-Ib' are cloned and purified
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His-tagged AAC(6')-Isa
on a His-Bind NiNTA column
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on a Ni-NTA and a Superdex S-75 column, the His6 tag is cleaved using thrombin
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recombinant AAC(6')-Iaf is purified using Ni-nitrilotriacetic acid agarose, native AAC(6')-Iaf from Pseudomonas aeruginosa on an anti-AAC(6')-Iaf-IgG coupled and NHS-activated Sepharose column
unseparable from 2-O-aminoglycoside phosphotransferase by affinity chromatography or sucrose density gradient ultracentrifugation
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
aac(6')-Ib gene
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aac(6')-Iz can restore aminoglycoside resistance to tobramycin, netilimicin and sisomicin (four- to eight-fold increase in the MICs) upon Escherichia coli
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both acetyltransferase domains of the bifunctional enzyme AAC(3)-Ib/AAC(6')-Ib' are cloned
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cloned into the pET23a(+) vector and overexpression in Escherichia coli conferrs high-level resistance to the usual substrates of aminoglycoside N-acetyltransferase except netilmicin
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expression in Escherichia coli
expression in Streptomyces lividans TK21/pANT-S2
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expression of mutant enzymes F171L and Y80C
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into the vector pET-28a+ for expression in Escherichia coli BL21DE3 cells
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into the vector pET19b
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into the vectors pSTV28 and pQE2, pQE2 is used for expression of the protein in Escherichia coli BL21DE3 pLysS cells
isogenic plasmids pSCH4663 containing aac(6')-Ib gene and pSCHB4105 containing aac(6')-IIa gene, transferred to Escherichia coli DH5alpha
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Pseudomonas aeruginosa 141, gene transfer via plasmid pBP30 to Escherichia coli Hb101
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
C-14T mutation in eis increases expression of aminoglycoside acetyltransferase eis
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E72A
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mutation causes a notable reduction in affinity for both the 4,5- and 4,5-disubstituted classes of aminoglycosides, with the largest changes being an 83fold decrease in the affinity for the minimal substrate neamine and a 175fold reduction in Km-value for kanamycin
H74A
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kinetic parameters reveal only a small effect of the mutation on enzyme activity, Km-values for acetyl-CoA and the 4,5- and 4,6-disubstituted aminoglycosides only differs by a range of 2-7.5fold compared to that of the wild-type enzyme, indicating no significant change in apparent affinity
L76A
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mutant enzyme shows virtually wild-type activity, with the biggest changes being a 3.6fold reduction in the apparent affinity for neomycin and a 3.7fold decrease in turnover number for amikacin
L76P
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mutant enzyme is impaired in both aminoglycoside recognition and catalysis, 2300fold decrease in the ratio of turnover number to Kb-value for neamine, 1300fold reduction in the ratio of turnover number to Kb-value for tobramycin, no detectable activity towardskanamycin A and amikacin
Y147A
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no detectable activity towardsamikacin
Y147F
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decrease in aminoglycoside affinity ranges from a 6fold decrease for tobramycin to a large 170fold change in KM-value for amikacin
D99A
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39fold decrease in the ratio of turnover number to Km-value for acetyl-CoA, 215fold decrease in the ratio of turnover number to Km-value for kanamycin A, 86fold decrease in the ratio of turnover number to Km-value for neamine
D99E
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4.7fold decrease in the ratio of turnover number to Km-value for acetyl-CoA, 31fold decrease in the ratio of turnover number to Km-value for kanamycin A, 19.3fold decrease in the ratio of turnover number to Km-value for neamine, 134fold decrease in the ratio of turnover number to Km-value for fortimicin A
D99N
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7fold decrease in the ratio of turnover number to Km-value for acetyl-CoA, 130fold decrease in the ratio of turnover number to Km-value for kanamycin A, 102fold decrease in the ratio of turnover number to Km-value for neamine
Y96F
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minor effects on the steady state kinetic parameters, largest effects for paromomycin, 5.5fold decrease in the ratio kcat /Km compared with wild-type
D117A
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complete loss of the resistance phenotype against kanamycin and amikacin
D120A
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complete loss of the resistance phenotype against kanamycin and amikacin
L119S
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AAC(6')-Ib L119S. The AAC(6')-Ib protein is unable to efficiently modify gentamicin C1, 1.7% relative to sisomicin, however it is capable of modifying amikacin, 65.5% relative to sisomycin. The mutation results in a 2.8fold increase in acetylation of gentamicin C1, but causes an 8.7fold reduction in the ability to modify amikacin
S119L
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AAC(6')-IIa S119L. The AAC(6')-IIa protein modifies gentamicin C1 at 10.1% relative to sisomicin, however it shows low activity towards amikacin, 4.1% relative to sisomycin. The mutation results in a 4.8fold reduction in the acetylation of gentamicin C1, but causes an 2fold increase in the ability to modify amikacin
C165A
mutant enzyme shows levels of resistance to both antibiotics no more than threefold lower than that for the wild type
E167A
highly reduced ability to confer resistance to kanamycin and amikacin
E172A
mutant enzyme shows levels of resistance to both antibiotics no more than threefold lower than that for the wild type
F171G
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the mutant enzyme is unable to confer resistance against amikacin, kanamycin, netilmicin and tobramicin
F171I
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the mutant enzyme shows reduced resistance against amikacin, kanamycin, netilmicin and tobramicin
F171K
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the mutant enzyme is unable to confer resistance against amikacin, kanamycin, netilmicin and tobramicin
F171M
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the mutant enzyme is able to confer detectable resistance against amikacin, kanamycin, netilmicin and tobramicin, although the levels are considerably lower than those conferred by the wild-type enzyme
F171N
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the mutant enzyme is unable to confer resistance against amikacin, kanamycin, netilmicin and tobramicin
F171S
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the mutant enzyme is unable to confer resistance against amikacin, kanamycin, netilmicin and tobramicin
F171W
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the mutant enzyme is able to confer detectable resistance against amikacin, kanamycin, netilmicin and tobramicin, although the levels are considerably lower than those conferred by the wild-type enzyme
G170A
mutant enzyme confers high-level resistance to kanamycin but loses the ability to confer resistance to amikacin
G175A
mutant enzyme shows levels of resistance to both antibiotics no more than threefold lower than that for the wild type
I163A
the percentage of loss of resistance to one of the two antibiotics, kanamycin and amikacin, is no more than twice the percentage of loss of resistance to the other antibiotic
K168A
the percentage of loss of resistance to one of the two antibiotics, kanamycin and amikacin, is no more than twice the percentage of loss of resistance to the other antibiotic
L160A
the percentage of loss of resistance to one of the two antibiotics, kanamycin and amikacin, is no more than twice the percentage of loss of resistance to the other antibiotic
N159A
mutant enzyme confers high-level resistance to kanamycin but loses the ability to confer resistance to amikacin
P155A
the percentage of loss of resistance to one of the two antibiotics, kanamycin and amikacin, is no more than twice the percentage of loss of resistance to the other antibiotic
P157A
the percentage of loss of resistance to one of the two antibiotics, kanamycin and amikacin, is no more than twice the percentage of loss of resistance to the other antibiotic
Q174A
mutant enzyme shows levels of resistance to both antibiotics no more than threefold lower than that for the wild type
R161A
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mutant enzyme shows levels of resistance to both antibiotics no more than threefold lower than that for the wild type
R164A
mutant enzyme shows levels of resistance to both antibiotics no more than threefold lower than that for the wild type
R173A
mutant enzyme shows levels of resistance to both antibiotics no more than threefold lower than that for the wild type
S156A
mutant enzyme shows levels of resistance to both antibiotics no more than threefold lower than that for the wild type
S158A
mutant enzyme shows levels of resistance to both antibiotics no more than threefold lower than that for the wild type; the MICs of amikacin and kanamycin are higher than those for the wild-type enzyme
Y166A
mutant enzyme confers high-level resistance to kanamycin but loses the ability to confer resistance to amikacin
Y80C
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the mutant enzyme shows only marginal levels of activity when either amikacin, kanamycin, tobramycin or netilmicin is used as substrate
C109A
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mutation neither abolishes activity nor alters the biphasic inactivation by iodoacetamide
C109A/C70A
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mutant enzyme is not inactivated by iodoacetamide. Double mutant exhibits large increases in Km-values for both acetyl-CoA and aminoglycoside substrates
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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