Information on EC 2.7.1.95 - kanamycin kinase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY
2.7.1.95
-
RECOMMENDED NAME
GeneOntology No.
kanamycin kinase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ATP + kanamycin = ADP + kanamycin 3'-phosphate
show the reaction diagram
-
-
-
-
ATP + kanamycin = ADP + kanamycin 3'-phosphate
show the reaction diagram
isoenzyme APH3'-IIIa follows a Theorell-Chance mechanism
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
phospho group transfer
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
ATP:kanamycin 3'-O-phosphotransferase
Also acts on the antibiotics neomycin, paromomycin, neamine, paromamine, vistamycin and gentamicin A. An enzyme from Pseudomonas aeruginosa also acts on butirosin.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
aminoglycoside phosphotransferase 3'-IIIa, i.e. APH3'-IIIa
-
-
aminoglycoside phosphotransferase VIII
-
-
aminoglycoside phosphotransferase VIII
Streptomyces rimosus ATCC 10970
-
-
-
APH
-
gene name
APH
Streptomyces rimosus ATCC 10970
-
gene name
-
aph(3'')-IIIa
-
gene name
kinase, kanamycin (phosphorylating)
-
-
-
-
neomycin phosphotransferase
-
-
-
-
neomycin phosphotransferase
-
-
neomycin phosphotransferase
-
-
neomycin-kanamycin phosphotransferase
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
62213-36-9
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
Escherichia coli strain transformed with plasmid encoding kanamycin kinase, higher antibiotica resistance in bacteria cultvated in the presence of putrescine
-
-
Manually annotated by BRENDA team
pKm2-transformed C-600 cells
-
-
Manually annotated by BRENDA team
R-factor strain JR35
-
-
Manually annotated by BRENDA team
strains JR35/W677, JR39/W677, JR66/W677, neomycin phosphotransferase II, ML1629, neomycin phosphotransferase I
-
-
Manually annotated by BRENDA team
strain Ps49, neomycin phosphotransferase II
-
-
Manually annotated by BRENDA team
ten strains: H1-H9 and A3
-
-
Manually annotated by BRENDA team
Pseudomonas aeruginosa Ps49
strain Ps49, neomycin phosphotransferase II
-
-
Manually annotated by BRENDA team
drug-resistant strain B295; multiple drug-resistant strain B294
-
-
Manually annotated by BRENDA team
multiple drug-resistant strain B294
-
-
Manually annotated by BRENDA team
Streptomyces rimosus ATCC 10970
-
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + kanamycin
ADP + kanamycin 3'-phosphate
show the reaction diagram
-
-
-
?
ATP + kanamycin
ADP + kanamycin 3'-phosphate
show the reaction diagram
-
-
-
-
ATP + kanamycin
ADP + kanamycin 3'-phosphate
show the reaction diagram
-
-
-
?
ATP + kanamycin
ADP + kanamycin 3'-phosphate
show the reaction diagram
-
-
-
?
ATP + kanamycin
ADP + kanamycin 3'-phosphate
show the reaction diagram
-
specific for ATP
-
?
ATP + kanamycin
ADP + kanamycin 3'-phosphate
show the reaction diagram
-
specific for ATP
-
?
ATP + kanamycin
ADP + kanamycin 3'-phosphate
show the reaction diagram
-
specific for ATP
-
?
ATP + kanamycin
ADP + kanamycin 3'-phosphate
show the reaction diagram
-
no activity with ADP, phosphorylates the C-3 hydroxyl group of 6-amino-6-deoxy-D-glucose moiety of kanamycin B or C
-
?
ATP + kanamycin
ADP + kanamycin 3'-phosphate
show the reaction diagram
-
no activity with streptomycin
-
?
ATP + kanamycin
ADP + kanamycin 3'-phosphate
show the reaction diagram
-
no activity with streptomycin
-
?
ATP + kanamycin
ADP + kanamycin 3'-phosphate
show the reaction diagram
-
isoenzyme APH3'-IIIa
-
r
ATP + kanamycin
ADP + kanamycin 3'-phosphate
show the reaction diagram
Pseudomonas aeruginosa Ps49
-
specific for ATP
-
?
ATP + neamine
ADP + neamine phosphate
show the reaction diagram
-
-
-
?
ATP + neomycin
ADP + neomyin phosphate
show the reaction diagram
-
-
-
?
ATP + neomycin
ADP + neomyin phosphate
show the reaction diagram
-
-
-
?
ATP + neomycin
ADP + neomyin phosphate
show the reaction diagram
-
-
-
?
ATP + neomycin
ADP + neomycin 3'-phosphate
show the reaction diagram
-
-
-
-
?
ATP + neomycin
ADP + neomycin 3'-phosphate
show the reaction diagram
-
-
-
-
-
ATP + neomycin
ADP + neomycin 3'-phosphate
show the reaction diagram
-
i.e. G418, specific inactivation of the antibiotic
-
-
?
ATP + paromomycin
ADP + paromomycin phosphate
show the reaction diagram
-
-
-
?
additional information
?
-
-
under physiologic concentrationsof NTPs, enzyme exclusively uses ATP
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + neomycin
ADP + neomycin 3'-phosphate
show the reaction diagram
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Co2+
-
activation at 1 mM, can replace Mg2+
Mg2+
-
activation
Mg2+
-
required for activity
Mn2+
-
activation at 1 mM, can replace Mg2+
Zn2+
-
activation at 1 mM, can replace Mg2+
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
Adenylyl imidodiphosphate
-
-
Gentamicin 1a
-
with neomycin as substrate, no inhibition in E. coli strain JR66/W677 or Pseudomonas aeruginosa strain Ps49
kanamycin
-
substrate inhibition at 2 mM
additional information
-
not inhibitory: guanylyl imidodiphosphate
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1.32
-
3'-phosphokanamycin
-
pH 7.5, 37C, isoenzyme APH3'-IIIa
-
0.022
-
ADP
-
pH 7.5, 37C, isoenzyme APH3'-IIIa
0.0106
-
ATP
-
pH 7.5, 37C
0.028
-
ATP
-
pH 7.5, 37C, isoenzyme APH3'-IIIa
0.3
-
kanamycin
-
pH 7.5, 35C
0.004
-
kanamycin A
-
pH 6.5, 37C, isoenzyme APH3'-IIIa
0.013
-
kanamycin A
-
pH 7.5, 37C, isoenzyme APH3'-IIIa
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.11
-
ATP
-
pH 7.5, 37C, isoenzyme APH3'-IIIa
1.59
-
ATP
-
pH 7.5, 37C
1.76
-
ATP
-
pH 7.5, 37C, isoenzyme APH3'-IIIa
1.79
-
kanamycin A
-
pH 7.5, 37C, isoenzyme APH3'-IIIa
kcat/KM VALUE [1/mMs-1]
kcat/KM VALUE [1/mMs-1] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
150
-
ATP
-
pH 7.5, 37C
22040
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0142
-
Adenylyl imidodiphosphate
-
pH 7.5, 37C
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.5
8
-
broad, no marked optimum
7.5
-
-
with kanamycin
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6
8
-
approx. half-maximal activity at pH 6.0 and pH 8.0
6.5
8
-
approx. half-maximal activity at pH 6.5 and pH 8.0
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
35
37
-
assay at
35
37
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
-
transformed with pAG60
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
phosphoprotein
-
residue Ser146 in the activation loop of the enzyme is the key site for Ca2+-dependent phosphorylation of isoform APHVIII. The Ser146 modification leads to a 6-7-fold increase in the kanamycin kinase activity of APHVII
phosphoprotein
Streptomyces rimosus ATCC 10970
-
residue Ser146 in the activation loop of the enzyme is the key site for Ca2+-dependent phosphorylation of isoform APHVIII. The Ser146 modification leads to a 6-7-fold increase in the kanamycin kinase activity of APHVII
-
pH STABILITY
pH STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
45
-
-
strain ML1629, 5 min, inactivation
55
-
-
strain H-9, 20 min stable
65
-
-
strain H-9, 5 min stable
75
-
-
strain H-9, inactivation
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ammonium chloride or neomycin B stabilizes, E. coli JR39 enzyme
-
dithiothreitol stabilizes and restores activity of denatured enzyme
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ammonium sulfate, Sephadex G 50, Sephadex G 100, partial purification
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expressed in COS-1 cells
-
expression of kanaR gene in Escherichia coli
-
expression of neomycin phosphotransferase II in Nicotiana tobacco
-
expression of neomycin phosphotransferase II-glycophorin A fusion protein in Escherichia coli and CHO cells
-
expression of neoR gene in Trypanosoma cruzi
-
overexpression of aminoglycoside phosphotransferase-IIIa, i.e. APH3'-IIIa in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
S146A
-
19% of the activity of phosphorylated wild-type, 90% of the activity of unphosphorylated wild-type, in presence of Ca2+
S146A/S160A
-
17% of the activity of phosphorylated wild-type, 85% of the activity of unphosphorylated wild-type, in presence of Ca2+
S146A/S160A/S215A
-
17% of the activity of phosphorylated wild-type, 60% of the activity of unphosphorylated wild-type, in presence of Ca2+
S215A
-
88% of the activity of phosphorylated wild-type, 67% of the activity of unphosphorylated wild-type, in presence of Ca2+
S95A/S160A
-
82% of the activity of phosphorylated wild-type, 95% of the activity of unphosphorylated wild-type, in presence of Ca2+
S146A
Streptomyces rimosus ATCC 10970
-
19% of the activity of phosphorylated wild-type, 90% of the activity of unphosphorylated wild-type, in presence of Ca2+
-
S146A/S160A
Streptomyces rimosus ATCC 10970
-
17% of the activity of phosphorylated wild-type, 85% of the activity of unphosphorylated wild-type, in presence of Ca2+
-
S146A/S160A/S215A
Streptomyces rimosus ATCC 10970
-
17% of the activity of phosphorylated wild-type, 60% of the activity of unphosphorylated wild-type, in presence of Ca2+
-
S215A
Streptomyces rimosus ATCC 10970
-
88% of the activity of phosphorylated wild-type, 67% of the activity of unphosphorylated wild-type, in presence of Ca2+
-
S95A/S160A
Streptomyces rimosus ATCC 10970
-
82% of the activity of phosphorylated wild-type, 95% of the activity of unphosphorylated wild-type, in presence of Ca2+
-
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
analysis
-
competitive assay that mimics in vivo nucleotide triphosphate concentrations and usage by the enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enables the determination of partitioning of phosphate flux from nucleotide triphosphate donors to antibiotics
medicine
-
the enzyme can be used in immunoliposome-mediated delivery for the lineage-specific selection of differentiated/committed stem cell progenies for transplantation, method development and evaluation, potential advantages over transfection with marker genes, fluorescence-activated and magnetic affinity cell-sorting, overview