BRENDA - Enzyme Database

Characterization of the bifunctional aminoglycoside-modifying enzyme ANT(3)-Ii/AAC(6)-IId from Serratia marcescens

Kim, C.; Hesek, D.; Zajicek, J.; Vakulenko, S.B.; Mobashery, S.; Biochemistry 45, 8368-8377 (2006)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
2.7.7.47
cloned in Escherichia coli BL21
Serratia marcescens
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
2.3.1.82
6'-N-acetylkanamycin A
competitive versus kanamycin A and noncompetitive/mixed versus acetyl-CoA
Serratia marcescens
2.3.1.82
butyryl-CoA
uncompetitive versus kanamycin A and competitive versus acetyl CoA
Serratia marcescens
2.3.1.82
CoASH
uncompetitive versus kanamycin A and noncompetitive/mixed versus acetyl-CoA
Serratia marcescens
2.3.1.82
paromomycin
competitive versus kanamycin A and noncompetitive/mixed versus acetyl-CoA
Serratia marcescens
2.7.7.47
9-O-(adenosine-5'-phophoryl)spectinomycin
product inhibition of ANT(3'')-Ii domain
Serratia marcescens
2.7.7.47
AMP-CPP
dead-end inhibition of ANT(3'')-Ii domain
Serratia marcescens
2.7.7.47
kanamycin A
dead-end inhibition of ANT(3'')-Ii domain
Serratia marcescens
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.3.1.82
0.012
-
kanamycin A
pH 7.5
Serratia marcescens
2.3.1.82
0.015
-
gentamicin
pH 7.5
Serratia marcescens
2.3.1.82
0.036
-
netilmicin
pH 7.5
Serratia marcescens
2.3.1.82
0.044
-
acetyl-CoA
pH 7.5
Serratia marcescens
2.3.1.82
0.313
-
amikacin
pH 7.5
Serratia marcescens
2.7.7.47
0.0013
-
streptomycin
kinetic parameter of the ANT(3'')-Ii domain of the enzyme
Serratia marcescens
2.7.7.47
0.0014
-
spectinomycin
kinetic parameter of the ANT(3'')-Ii domain of the enzyme
Serratia marcescens
2.7.7.47
0.014
-
ATP
kinetic parameter of the ANT(3'')-Ii domain of the enzyme
Serratia marcescens
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
2.7.7.47
Mg2+
assay with 15 mM MgCl2
Serratia marcescens
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
2.3.1.82
Serratia marcescens
-
-
-
2.7.7.47
Serratia marcescens
-
-
-
Purification (Commentary)
EC Number
Commentary
Organism
2.7.7.47
DEAE anion-exchange column and gentamycin affinity column
Serratia marcescens
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.3.1.82
acetyl-CoA + amikacin
-
672194
Serratia marcescens
CoA + N6'-acetylamikacin
-
-
-
?
2.3.1.82
acetyl-CoA + gentamicin
-
672194
Serratia marcescens
CoA + N6'-acetylgentamicin
-
-
-
?
2.3.1.82
acetyl-CoA + kanamycin A
the bifunctional antibiotic resistance enzyme from Serratia marcescens catalyzes adenylation and acetylation of aminoglycoside antibiotics. The structure assignment of the enzymic products indicated that acetylation takes place on the 6'-amine of kanamycin A and the adenylation on 3''- and 9-hydroxyl groups of streptomycin and spectinomycin, respectively. The adenyltransferase domain appears to be highly specific to spectinomycin and streptomycin, while the acetyltransferase domain shows a broad substrate profile. Initial velocity patterns indicate that both domains follow a sequential kinetic mechanism
672194
Serratia marcescens
CoA + N6'-acetylkanamycin A
-
-
-
?
2.3.1.82
acetyl-CoA + netilmicin
-
672194
Serratia marcescens
CoA + N6'-acetylnetilmicin
-
-
-
?
2.7.7.47
ATP + spectinomycin
-
672194
Serratia marcescens
diphosphate + 9-adenylylspectinomycin
-
-
-
-
2.7.7.47
ATP + streptomycin
bifunctional enzyme, adenylation of aminoglycoside antibiotics takes place at the ANT(3'')-Ii domain, aceylation of aminoglycoside antibiotics takes place at the AAC(6')-domain
672194
Serratia marcescens
diphosphate + 3''-adenylylstreptomycin
-
-
-
?
2.7.7.47
additional information
adenyltransferase domain is highly specific for spectinomycin and streptomycin and catalyzes the reaction by a Theorell-Chance kinetic mechanism, where ATP binds to the enzyme prior to the aminoglycoside and the modified antibiotic is the last product to be released
672194
Serratia marcescens
?
-
-
-
-
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
2.7.7.47
additional information
-
assay is performed at room temperature
Serratia marcescens
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.3.1.82
0.05
-
amikacin
pH 7.5
Serratia marcescens
2.3.1.82
0.2
-
netilmicin
pH 7.5
Serratia marcescens
2.3.1.82
0.5
-
gentamicin
pH 7.5
Serratia marcescens
2.3.1.82
0.8
-
acetyl-CoA
pH 7.5
Serratia marcescens
2.3.1.82
0.8
-
kanamycin A
pH 7.5
Serratia marcescens
2.7.7.47
0.5
-
ATP
turnover number of ANT(3'')-Ii domain
Serratia marcescens
2.7.7.47
0.5
-
spectinomycin
turnover number of ANT(3'')-Ii domain
Serratia marcescens
2.7.7.47
0.6
-
streptomycin
turnover number of ANT(3'')-Ii domain
Serratia marcescens
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
2.7.7.47
7.5
-
assay at
Serratia marcescens
Ki Value [mM]
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
2.3.1.82
0.025
-
butyryl-CoA
versus acetyl-CoA, K(is)
Serratia marcescens
2.3.1.82
0.04
-
6'-N-acetylkanamycin A
versus kanamycin A, K(is)
Serratia marcescens
2.3.1.82
0.042
-
CoASH
versus acetyl-CoA, K(is)
Serratia marcescens
2.3.1.82
0.043
-
CoASH
versus acetyl-CoA, K(ii)
Serratia marcescens
2.3.1.82
0.06
-
paromomycin
versus kanamycin A, K(is)
Serratia marcescens
2.3.1.82
0.069
-
CoASH
versus kanamycin A, K(ii)
Serratia marcescens
2.3.1.82
0.117
-
paromomycin
versus acetyl-CoA, K(is)
Serratia marcescens
2.3.1.82
0.145
-
paromomycin
versus acetyl-CoA, K(ii)
Serratia marcescens
2.3.1.82
0.15
-
6'-N-acetylkanamycin A
versus acetyl-CoA, K(is)
Serratia marcescens
2.3.1.82
0.188
-
butyryl-CoA
versus kanamycin A, K(ii)
Serratia marcescens
2.3.1.82
0.385
-
6'-N-acetylkanamycin A
versus acetyl-CoA, K(ii)
Serratia marcescens
2.7.7.47
0.004
-
kanamycin A
dead-end inhibition of ANT(3'')-Ii domain, substrate 0.02 mM spectinomycin, competitive inhibition
Serratia marcescens
2.7.7.47
0.01
-
kanamycin A
Kii = 0.01 mM, dead-end inhibition of ANT(3'')-Ii domain, substrate 0.1 mM ATP, uncompetitive inhibition
Serratia marcescens
2.7.7.47
0.017
-
AMP-CPP
dead-end inhibition of ANT(3'')-Ii domain, substrate 0.1 mM ATP, competitive inhibition
Serratia marcescens
2.7.7.47
0.021
-
AMP-CPP
dead-end inhibition of ANT(3'')-Ii domain, substrate 0.02 mM spectinomycin, Kii = 0.031 mM, noncompetitive/mixed inhibition
Serratia marcescens
2.7.7.47
0.076
-
9-O-(adenosine-5'-phophoryl)spectinomycin
Kii = 0.089, product inhibition of ANT(3'')-Ii domain, substrate 0.02 mM spectinomycin, noncompetitive/mixed inhibition
Serratia marcescens
2.7.7.47
0.093
-
9-O-(adenosine-5'-phophoryl)spectinomycin
product inhibition of ANT(3'')-Ii domain, substrate 0.1 mM ATP, competitive inhibition
Serratia marcescens
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
2.7.7.47
cloned in Escherichia coli BL21
Serratia marcescens
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
2.3.1.82
6'-N-acetylkanamycin A
competitive versus kanamycin A and noncompetitive/mixed versus acetyl-CoA
Serratia marcescens
2.3.1.82
butyryl-CoA
uncompetitive versus kanamycin A and competitive versus acetyl CoA
Serratia marcescens
2.3.1.82
CoASH
uncompetitive versus kanamycin A and noncompetitive/mixed versus acetyl-CoA
Serratia marcescens
2.3.1.82
paromomycin
competitive versus kanamycin A and noncompetitive/mixed versus acetyl-CoA
Serratia marcescens
2.7.7.47
9-O-(adenosine-5'-phophoryl)spectinomycin
product inhibition of ANT(3'')-Ii domain
Serratia marcescens
2.7.7.47
AMP-CPP
dead-end inhibition of ANT(3'')-Ii domain
Serratia marcescens
2.7.7.47
kanamycin A
dead-end inhibition of ANT(3'')-Ii domain
Serratia marcescens
Ki Value [mM] (protein specific)
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
2.3.1.82
0.025
-
butyryl-CoA
versus acetyl-CoA, K(is)
Serratia marcescens
2.3.1.82
0.04
-
6'-N-acetylkanamycin A
versus kanamycin A, K(is)
Serratia marcescens
2.3.1.82
0.042
-
CoASH
versus acetyl-CoA, K(is)
Serratia marcescens
2.3.1.82
0.043
-
CoASH
versus acetyl-CoA, K(ii)
Serratia marcescens
2.3.1.82
0.06
-
paromomycin
versus kanamycin A, K(is)
Serratia marcescens
2.3.1.82
0.069
-
CoASH
versus kanamycin A, K(ii)
Serratia marcescens
2.3.1.82
0.117
-
paromomycin
versus acetyl-CoA, K(is)
Serratia marcescens
2.3.1.82
0.145
-
paromomycin
versus acetyl-CoA, K(ii)
Serratia marcescens
2.3.1.82
0.15
-
6'-N-acetylkanamycin A
versus acetyl-CoA, K(is)
Serratia marcescens
2.3.1.82
0.188
-
butyryl-CoA
versus kanamycin A, K(ii)
Serratia marcescens
2.3.1.82
0.385
-
6'-N-acetylkanamycin A
versus acetyl-CoA, K(ii)
Serratia marcescens
2.7.7.47
0.004
-
kanamycin A
dead-end inhibition of ANT(3'')-Ii domain, substrate 0.02 mM spectinomycin, competitive inhibition
Serratia marcescens
2.7.7.47
0.01
-
kanamycin A
Kii = 0.01 mM, dead-end inhibition of ANT(3'')-Ii domain, substrate 0.1 mM ATP, uncompetitive inhibition
Serratia marcescens
2.7.7.47
0.017
-
AMP-CPP
dead-end inhibition of ANT(3'')-Ii domain, substrate 0.1 mM ATP, competitive inhibition
Serratia marcescens
2.7.7.47
0.021
-
AMP-CPP
dead-end inhibition of ANT(3'')-Ii domain, substrate 0.02 mM spectinomycin, Kii = 0.031 mM, noncompetitive/mixed inhibition
Serratia marcescens
2.7.7.47
0.076
-
9-O-(adenosine-5'-phophoryl)spectinomycin
Kii = 0.089, product inhibition of ANT(3'')-Ii domain, substrate 0.02 mM spectinomycin, noncompetitive/mixed inhibition
Serratia marcescens
2.7.7.47
0.093
-
9-O-(adenosine-5'-phophoryl)spectinomycin
product inhibition of ANT(3'')-Ii domain, substrate 0.1 mM ATP, competitive inhibition
Serratia marcescens
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.3.1.82
0.012
-
kanamycin A
pH 7.5
Serratia marcescens
2.3.1.82
0.015
-
gentamicin
pH 7.5
Serratia marcescens
2.3.1.82
0.036
-
netilmicin
pH 7.5
Serratia marcescens
2.3.1.82
0.044
-
acetyl-CoA
pH 7.5
Serratia marcescens
2.3.1.82
0.313
-
amikacin
pH 7.5
Serratia marcescens
2.7.7.47
0.0013
-
streptomycin
kinetic parameter of the ANT(3'')-Ii domain of the enzyme
Serratia marcescens
2.7.7.47
0.0014
-
spectinomycin
kinetic parameter of the ANT(3'')-Ii domain of the enzyme
Serratia marcescens
2.7.7.47
0.014
-
ATP
kinetic parameter of the ANT(3'')-Ii domain of the enzyme
Serratia marcescens
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
2.7.7.47
Mg2+
assay with 15 mM MgCl2
Serratia marcescens
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
2.7.7.47
DEAE anion-exchange column and gentamycin affinity column
Serratia marcescens
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.3.1.82
acetyl-CoA + amikacin
-
672194
Serratia marcescens
CoA + N6'-acetylamikacin
-
-
-
?
2.3.1.82
acetyl-CoA + gentamicin
-
672194
Serratia marcescens
CoA + N6'-acetylgentamicin
-
-
-
?
2.3.1.82
acetyl-CoA + kanamycin A
the bifunctional antibiotic resistance enzyme from Serratia marcescens catalyzes adenylation and acetylation of aminoglycoside antibiotics. The structure assignment of the enzymic products indicated that acetylation takes place on the 6'-amine of kanamycin A and the adenylation on 3''- and 9-hydroxyl groups of streptomycin and spectinomycin, respectively. The adenyltransferase domain appears to be highly specific to spectinomycin and streptomycin, while the acetyltransferase domain shows a broad substrate profile. Initial velocity patterns indicate that both domains follow a sequential kinetic mechanism
672194
Serratia marcescens
CoA + N6'-acetylkanamycin A
-
-
-
?
2.3.1.82
acetyl-CoA + netilmicin
-
672194
Serratia marcescens
CoA + N6'-acetylnetilmicin
-
-
-
?
2.7.7.47
ATP + spectinomycin
-
672194
Serratia marcescens
diphosphate + 9-adenylylspectinomycin
-
-
-
-
2.7.7.47
ATP + streptomycin
bifunctional enzyme, adenylation of aminoglycoside antibiotics takes place at the ANT(3'')-Ii domain, aceylation of aminoglycoside antibiotics takes place at the AAC(6')-domain
672194
Serratia marcescens
diphosphate + 3''-adenylylstreptomycin
-
-
-
?
2.7.7.47
additional information
adenyltransferase domain is highly specific for spectinomycin and streptomycin and catalyzes the reaction by a Theorell-Chance kinetic mechanism, where ATP binds to the enzyme prior to the aminoglycoside and the modified antibiotic is the last product to be released
672194
Serratia marcescens
?
-
-
-
-
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
2.7.7.47
additional information
-
assay is performed at room temperature
Serratia marcescens
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.3.1.82
0.05
-
amikacin
pH 7.5
Serratia marcescens
2.3.1.82
0.2
-
netilmicin
pH 7.5
Serratia marcescens
2.3.1.82
0.5
-
gentamicin
pH 7.5
Serratia marcescens
2.3.1.82
0.8
-
acetyl-CoA
pH 7.5
Serratia marcescens
2.3.1.82
0.8
-
kanamycin A
pH 7.5
Serratia marcescens
2.7.7.47
0.5
-
ATP
turnover number of ANT(3'')-Ii domain
Serratia marcescens
2.7.7.47
0.5
-
spectinomycin
turnover number of ANT(3'')-Ii domain
Serratia marcescens
2.7.7.47
0.6
-
streptomycin
turnover number of ANT(3'')-Ii domain
Serratia marcescens
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
2.7.7.47
7.5
-
assay at
Serratia marcescens