Any feedback?
Information on EC 2.7.1.190 - aminoglycoside 2''-phosphotransferase
for references in articles please use BRENDA:EC2.7.1.190Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
EC Tree
2.7.1.190 aminoglycoside 2''-phosphotransferaseIUBMB Comments
Requires Mg2+. This bacterial enzyme phosphorylates many 4,6-disubstituted aminoglycoside antibiotics that have a hydroxyl group at position 2'', including kanamycin A, kanamycin B, tobramycin, dibekacin, arbekacin, amikacin, gentamicin C, sisomicin and netilmicin. In most, but not all, cases the phosphorylation confers resistance against the antibiotic. Some forms of the enzyme use ATP as a phosphate donor in appreciable amount. The enzyme is often found as a bifunctional enzyme that also catalyses 6'-aminoglycoside N-acetyltransferase activity. The bifunctional enzyme is the most clinically important aminoglycoside-modifying enzyme in Gram-positive bacteria, responsible for high-level resistance in both Enterococci and Staphylococci.
Specify your search results
Word Map
- 2.7.1.190
-
aph3\'-iiia
- kanamycin
-
enterococci
-
aac6\'-ie-aph2\'\'-ia
- faecium
-
aminoglycoside-modifying
-
phosphotransferases
-
aph2\'\'-ic
- amikacin
-
gentamicin-resistant
-
aac6\'-aph2
- tobramycin
-
4,6-disubstituted
- casseliflavus
-
aac6'-ie
-
ant6\'-ia
- gallinarum
-
aminoglycoside-resistance
- lividomycin
- butirosin
-
o-phosphorylation
- sisomicin
- durans
- molecular biology
The enzyme appears in viruses and cellular organisms
Synonyms
aph(2'')-id, aph(2''), aminoglycoside kinase, aph(2'')-ia, aph(2'')-iva, aac6-aph2, aph(2'')-iiia, aminoglycoside 2''-phosphotransferase, aminoglycoside 2''-phosphotransferase iva, aminoglycoside 2''-phosphotransferase type iiia, 
more
topPlease wait a moment until the data is sorted. This message will disappear when the data is sorted.
SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
aacA-aphD
aminoglycoside (2'') kinase
-
-
-
-
aminoglycoside phosphotransferase
APH
APH(2'')
APH(2'')-IVa
aphD
-
-
-
-
AphSR2
bifunctional 6'-aminoglycoside acetyltransferase/2"-aminoglycoside phosphotransferase
bifunctional AAC/APH
bifunctional aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2'')-Ia
-
bifunctional aminoglycoside acetyltransferase/phosphotransferase
-
gentamicin kinase
-
-
ambiguous
-
gentamicin phosphotransferase
-
-
ambiguous
-
APH(2'')
-
-
-
-
bifunctional 6'-aminoglycoside acetyltransferase/2"-aminoglycoside phosphotransferase
-
bifunctional 6'-aminoglycoside acetyltransferase/2"-aminoglycoside phosphotransferase
-
-
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
GTP + gentamicin = GDP + gentamicin 2''-phosphate
-
-
-
-
GTP + gentamicin = GDP + gentamicin 2''-phosphate
mechanism of the phosphorylation of aminoglycoside antibiotics by APH(2'') enzymes, detailed overview
-
GTP + gentamicin = GDP + gentamicin 2''-phosphate
the reaction of APH(2'')-IVa proceeds by a Bi-Bi mechanism in which aminoglycoside and ATP or GTP bind randomly
-
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
SYSTEMATIC NAME
IUBMB Comments
GTP:gentamicin 2''-O-phosphotransferase
Requires Mg2+. This bacterial enzyme phosphorylates many 4,6-disubstituted aminoglycoside antibiotics that have a hydroxyl group at position 2'', including kanamycin A, kanamycin B, tobramycin, dibekacin, arbekacin, amikacin, gentamicin C, sisomicin and netilmicin. In most, but not all, cases the phosphorylation confers resistance against the antibiotic. Some forms of the enzyme use ATP as a phosphate donor in appreciable amount. The enzyme is often found as a bifunctional enzyme that also catalyses 6'-aminoglycoside N-acetyltransferase activity. The bifunctional enzyme is the most clinically important aminoglycoside-modifying enzyme in Gram-positive bacteria, responsible for high-level resistance in both Enterococci and Staphylococci.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + gentamicin
ADP + gentamicin 2''-phosphate
-
GTP is preferred over ATP
-
?
GTP + gentamicin C
GDP + gentamicin C 2''-phosphate
-
GTP is preferred over ATP
-
?
GTP + gentamicin C
GDP + gentamicin C 2''-phosphate
-
-
GTP is preferred over ATP
-
?
GTP + kanamycin A
GDP + kanamycin A 2''-phosphate
-
-
-
?
GTP + lividomycin
GDP + lividomycin 2''-phosphate
-
enzyme binding structure analysis, overview
-
-
?
additional information
?
-
no substrates: 4,5-disubstituted antibiotics and the atypical aminoglycoside neamine
-
-
?
additional information
?
-
-
no substrates: 4,5-disubstituted antibiotics and the atypical aminoglycoside neamine
-
-
?
additional information
?
-
-
enzyme assay with coupling the release of ADP to a pyruvate kinase/lactate dehydrogenase reaction
-
-
-
additional information
?
-
-
most aminoglycosides are based upon a neamine core, i.e. a central 2-deoxystreptamine (2-DOS) ring with an aminohexose sugar linked to 2-DOS at the 4-position. These rings form the minimal active element of an aminoglycoside antibiotic, and two subclasses of aminoglycosides are formed from elaboration of this scaffold. Addition of rings to the 5- and 6-positions of 2-DOS differentiate aminoglycosides into the 4,5-disubstituted (ribostamycin, neomycin, and lividomycin) and 4,6-disusbstituted (kanamycin, gentamicin, and dibekacin) groups
-
-
-
additional information
?
-
-
random sequential Bi Bi mechanism. At pH 7.5 the release of guanosine triphosphate is rate-limiting. No substrates: amikacin, isepamicin, neomycin, butirosin, lividomycin A , paromomycin, spectinomycin, streptomycin, apramycin, hygromycin, neamine
-
-
?
additional information
?
-
-
substrate binding in the APH(2'') enzymes, overview. The substrate molecules are bound in essentially the same orientation in all structures. The neamine moiety of aminoglycosides is nestled against the core subdomain and helix alpha9 such that the A ring projects towards motif 3 in the helical subdomain and the B ring points towards motif 1 and motif 2 in the core subdomain. The C ring then projects back towards the helical domain
-
-
-
additional information
?
-
GTP is the exclusive phosphate donor at intracellular nucleotide levels
-
-
?
additional information
?
-
the bifunctional enzyme AAC(6')-APH(2'') enzyme shows acetyltransferase activity and phosphotransferase activity, activity measurement in a sigle assay. GTP is more qualified as a phosphate donor than ATP to cause phosphorylation events of AAC(6')-APH(2'')
-
-
-
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
GTP is the exclusive phosphate donor at intracellular nucleotide levels
-
-
?
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
cofactor binding structures, conformations of triphosphate group in structures of APH(2'')-Ia with GTP, GDP, and GMPPNP, overview. Two hydrogen bonds are formed between the triphosphate and residues S214 of the Gly loop and Y237. The nucleoside cosubstrates bind in the cleft between the N lobe (residues 180-279) and C lobe (residues 280-479), and two magnesium ions, Mg1 and Mg2, are consistently resolved with coordinating water molecules
-
additional information
-
cofactor binding structures, conformations of triphosphate group in structures of APH(2'')-Ia with GTP, GDP, and GMPPNP, overview. Two hydrogen bonds are formed between the triphosphate and residues S214 of the Gly loop and Y237. The nucleoside cosubstrates bind in the cleft between the N lobe (residues 180-279) and C lobe (residues 280-479), and two magnesium ions, Mg1 and Mg2, are consistently resolved with coordinating water molecules
-
additional information
GTP is more qualified as a phosphate donor than ATP to cause phosphorylation events of AAC(6')-APH(2'')
-
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Mg2+
required, magnesium ions, Mg1 and Mg2, are consistently resolved with coordinating water molecules
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ZINC13236811
-
63% inhibition at 0.5 mM of APH(2'')-IVa, ZINC13236811 is a non-competitive inhibitor of APH(2'')-IVa towards both ATP and ZINC8790387
additional information
-
identification os specific inhibitors of APHs that can restore bacterial susceptibility to aminoglycosides, screening of the Zinc database for search for allosteric inhibitors that bind to small cavities of the protein and block the enzyme function by perturbing its dynamics, docking study usig the crystal structure of the enzyme (PDB ID 3SG8) with removed aminoglycoside substrate, overview. The two interactions with side chain of Arg211 and backbones of Leu209 and Gly210 are shown to play an essential role in the inhibition of APH. No inhibition by ZINC6751595 and ZINC8981627
-
additional information
-
the ternary complex between APH(2'')-IIIa, GDP and kanamycin can be regarded as an inactive abortive complex, since the gamma-phosphate group which would normally be transferred to the 2''-hydroxyl of the substrate is absent. The cofactor binding in the ternary complex is similar in detail to that in the previously described binary complex
-
additional information
inhibitor screening of a chemical library using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry, assay method optimization and validation, overview. Poor inhibition by Mer-C-3, Mer-C-8e, and paromomycin
-
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
DISEASE
TITLE OF PUBLICATION
LINK TO PUBMED
Infections
Characterization of Aminoglycoside Resistance and Virulence Genes among Enterococcus spp. Isolated from a Hospital in China.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.22
ATP
mutant Y92A, pH not specified in the publication, temperature not specified in the publication
1.6
ATP
wild-type, pH not specified in the publication, temperature not specified in the publication
0.04
GTP
wild-type, pH not specified in the publication, temperature not specified in the publication
0.094
GTP
mutant Y92A, pH not specified in the publication, temperature not specified in the publication
additional information
additional information
-
steady-state kinetics
-
additional information
additional information
Michaelis-Menten kinetics
-
additional information
additional information
-
steady-state and transient kinetics
-
additional information
additional information
-
steady-state and transient kinetics
-
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.074
ZINC13236811
-
pH 7.5, 25°C, recombinant enzyme, versus kanamycin
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
isoform aminoglycoside-2''-phosphotransferase type IVa
UniProt
brenda
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
-
if the enzyme's binding mode is made impossible because of additional substitutions to the standard 4,5- or 4,6-disubstituted aminoglycoside architecture, as in lividomycin A or the N1-substituted aminoglycosides, it is still possible for these aminoglycosides to bind to the antibiotic binding site by using alternate binding modes, which explains the low rates of noncanonical phosphorylation activities seen in enzyme assays. A clinically observed arbekacin-resistant mutant of APH(2'')-Ia reveals an altered aminoglycoside binding site that can stabilize an alternative binding mode for N1-substituted aminoglycosides. This mutation may alter and expand the aminoglycoside resistance spectrum of the wild-type enzyme in response to developed aminoglycosides
physiological function
additional information
evolution
-
aminoglycoside phosphotransferases (APHs) are one of three families of aminoglycoside-modifying enzymes that confer high-level resistance to the aminoglycoside antibiotics via enzymatic modification, the APH(2'') family comprises four distinct members
evolution
-
Streptomyces rimosus ATCC 10970 contains 14 genes annotated as aminoglycoside phosphotransferases in its genome: aphSR1-aphSR14
evolution
-
Streptomyces rimosus ATCC 10970 contains 14 genes annotated as aminoglycoside phosphotransferases in its genome: aphSR1-aphSR14
-
physiological function
enzyme phosphorylates 4,6-disubstituted aminoglycosides with high efficiency. Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. Phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme
physiological function
expression in Escherichia coli confers resistance to the 4,6-disubstituted aminoglycosides kanamycin, tobramycin, dibekacin, gentamicin, and sisomicin, but not to arbekacin, amikacin, isepamicin, or netilmicin, but not to any of the 4,5-disubstituted antibiotics tested
physiological function
APH(2'')-Ia is a widely disseminated resistance factor frequently found in clinical isolates of Staphylococcus aureus and pathogenic enterococci, where it is constitutively expressed. APH(2'')-Ia confers high-level resistance to gentamicin and related aminoglycosides through phosphorylation of the antibiotic using GTP as phosphate donor
physiological function
-
change in the level of resistance to aminoglycoside antibiotics upon combined expression of the aphSR2, pkSR1, and pkSR2 genes in Escherichia coli strain BL21(DE3), overview
physiological function
-
the APH(2'')-Ia aminoglycoside resistance enzyme forms the C-terminal domain of the bifunctional AAC(6')-Ie/APH(2'')-Ia enzyme and confers high-level resistance to natural 4,6-disubstituted aminoglycosides. The enzyme can phosphorylate 4,5-disubstituted compounds and aminoglycosides with substitutions at the N1 position
physiological function
the bacterial enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2'')-Ia possesses an N-terminal acetyltransferase domain and a C-terminal phosphotransferase domain that can act synergistically and detoxify aminoglycoside antibiotics highly efficiently
physiological function
-
change in the level of resistance to aminoglycoside antibiotics upon combined expression of the aphSR2, pkSR1, and pkSR2 genes in Escherichia coli strain BL21(DE3), overview
-
additional information
-
APH(2'')-Ia maintains a preferred mode of binding aminoglycosides by using the conserved neamine rings when possible, with flexibility that allows it to accommodate additional rings
additional information
-
coexpression of aphSR2 gene and genes pkSR1 and pkSR2, encoding serine-threonine protein kinases, causes a 2fold increase in resistance to neomycin
additional information
-
structural basis for the diversity of the mechanism of nucleotide hydrolysis by the aminoglycoside-2''-phosphotransferases. Structure comparisons of the ternary complex of APH(2'')-IIIa with GDP and kanamycin with substrate-bound structures of APH(2'')-Ia, APH(2'')-IIa and APH(2'')-IVa. In contrast to the case for APH(2'')-Ia, where it was proposed that the enzyme-mediated hydrolysis of GTP is regulated by conformational changes in its N-terminal domain upon GTP binding, APH(2'')-IIa, APH(2'')-IIIa and APH(2'')-IVa show no such regulatory mechanism, primarily owing to structural differences in the N-terminal domains of these enzymes. The ternary complex between APH(2'')-IIIa, GDP and kanamycin can be regarded as an inactive abortive complex, since the gamma-phosphate group which would normally be transferred to the 2''-hydroxyl of the substrate is absent. The cofactor binding in the ternary complex is similar in detail to that in the previously described binary complex
additional information
the open-closed transition in APH(2'')-Ia brings distal regions of the protein into contact. The enzyme also exhibits a novel phenomenon: a switch between two welldefined triphosphate conformations. Interactions between the helical subdomain and N lobe loops connect enzyme closure to triphosphate activation. APH(2'')-Ia open-closed transition links aminoglycoside binding to catalysis through the Gly loop. In the stabilized conformation, the enzyme does not form most of the interactions that are required for catalysis in kinases. The gamma-phosphate does not coordinate between the two catalytic magnesium ions. There is no catalytic base in position to activate the incoming nucleophile, and no positively charged residue in place to stabilize the leaving group. To hydrogen bonds are formed between the triphosphate and residues S214 of the Gly oop and Y237. Aminoglycoside molecules bind in the cleft between the core (residues 280-322 and 366-432) and helical (residues 322-365 and 433-479) subdomains of the C lobe, the nucleoside cosubstrates bind in the cleft between the N lobe (residues 180-279) and C lobe (residues 280-479), and two magnesium ions, Mg1 and Mg2, are consistently resolved with coordinating water molecules. Aminoglycosides bind to APH(2'')-Ia via conserved rings, while variable rings Dictate reactivity
additional information
-
the open-closed transition in APH(2'')-Ia brings distal regions of the protein into contact. The enzyme also exhibits a novel phenomenon: a switch between two welldefined triphosphate conformations. Interactions between the helical subdomain and N lobe loops connect enzyme closure to triphosphate activation. APH(2'')-Ia open-closed transition links aminoglycoside binding to catalysis through the Gly loop. In the stabilized conformation, the enzyme does not form most of the interactions that are required for catalysis in kinases. The gamma-phosphate does not coordinate between the two catalytic magnesium ions. There is no catalytic base in position to activate the incoming nucleophile, and no positively charged residue in place to stabilize the leaving group. To hydrogen bonds are formed between the triphosphate and residues S214 of the Gly oop and Y237. Aminoglycoside molecules bind in the cleft between the core (residues 280-322 and 366-432) and helical (residues 322-365 and 433-479) subdomains of the C lobe, the nucleoside cosubstrates bind in the cleft between the N lobe (residues 180-279) and C lobe (residues 280-479), and two magnesium ions, Mg1 and Mg2, are consistently resolved with coordinating water molecules. Aminoglycosides bind to APH(2'')-Ia via conserved rings, while variable rings Dictate reactivity
additional information
-
coexpression of aphSR2 gene and genes pkSR1 and pkSR2, encoding serine-threonine protein kinases, causes a 2fold increase in resistance to neomycin
-
Show AA Sequence and Transmembrane Helices (264 entries)
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
PDB
SCOP
CATH
UNIPROT
ORGANISM
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
apo form and in complex with a bound antibiotic, tobramycin and kanamycin A, to 2.05 A, 1.8 A and 2.15 A resolution, respectively. Substrate binding induces conformational changes, involving rotational shifts of two distinct segments of the enzyme. The helical subdomain is important in substrate specificity
crystal structures of isoform APH(2'')-IVa, wild-type and mutant F95M, in complex with either adenosine or guanosine
enzyme APH(2'')-Ia crystals, grown with guanosine-beta,gamma-imidotriphosphate (GMPPNP) and a saturating concentration of magnesium, are soaked with ribostamycin, hanging drop vapour diffusion method, mixing of 0.001 ml ofp protein solution with 0.001 ml of reservoir solution containing 100 mM HEPES, pH 7.5, 120 mM MgCl2, 10% PEG 3350, and 10% glycerol, preincubation of the enzyme with 3 mM GMPPNP and 6 mM MgCl2, X-ray diffraction structure determination and analysis at 2.20-2.60 A resolution, model building
-
purified enzyme in complex with substrate G418 and sisomicin, mixing 0.001 ml of 6 mg/ml protein and 2.5 mM aminoglycoside in 50 mM HEPES, pH 7.5, and 10 mM MgCl2 with 0.001 ml of reservoir solution containing 12% PEG 3350 w/v, and 50-75 mM ammonium citrate, pH 7.4-7.9, 18°C, X-ray diffraction structure determination and analysis at 3.05 A and 2.35 A resolution, respectively, molecular replacement using chain A of the homologous APH(2'')-IVa-ADP complex (PDB ID 4N57) after removal of the ligand as search model, modeling
-
to 2.2 A resolution. Access to the ATP-binding template is blocked by a bulky tyrosine residue. Substitution of this tyrosine by a smaller amino acid opens access to the ATP template
ternary complex of APH(2'')-IIIa with GDP and kanamycin, the ternary complex s prepared by adding a tenfold molar excess of Mg2-GTP and kanamycin to the apo APH(2'')-IIIa F108L enzyme, followed by incubation of the complex at 4x02C for 2 h, the complex is crystallized from 30% PEG 4000, and 0.1 M Tris-HCl pH 8.5, X-ray diffraction structure determination at 1.34 A resolution, molecular replacement using the binary Mg2-GDP-APH(2'')-IIIa complex as the starting model (PDB ID 3tdw)
-
purified recombinant wild-type and mutant enzymes in complex with cofactor GTP, GDP, and especially GMPPNP, and substrates gentamycin C1, rbiostamycin, kanamycin A, neomycin B in diffenrent combinations, X-ray diffraction structure determination and analysis at 2.15-2.50 A resolution
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
F95M
no significantly different binding affinities as compared with the wild-type
F95Y
mutation shifts the nucleotide selectivity from a 2.5fold preference for ATP to a 1.5fold preference for GTP
S376N
-
a clinically identified naturally occuring S376N mutation of APH(2'')-Ia elevates resistance to N1-substituted aminoglycosides but eliminates modification of nonsubstituted compounds. Mutation of serine 376 to asparagine does not lead to substantial rearrangements in the aminoglycoside binding site. In fact, the addition of the larger asparagine residue in place of serine 376 creates an obstruction that prevents binding in the neamine binding pocket. As a result, any compounds that bind using the neamine rings are blocked from the antibiotic binding site of APH(2x02)-Ia. But the mutation remains compatible with one of the alternate binding modes of amikacin, which does not use this site to interact with the enzyme
Y92A
residue Y92 blocks access to the ATP-binding template. Mutant shows 8fold decrease in km value for ATP
F108L
-
site-directed mutagenesis, the point mutant and wild-type enzymes have the same structure
Y237F
site-directed mutagenesis, the mutant removes the other hydrogen bond between the phosphate and the protein, and a greatly reduced electron density for the gamma-phosphate is observed
additional information
additional information
The N-terminal region contains a sequence homologous to the chloramphenicol acetyltransferase of Bacillus pumUus, and the C-terminal region contains a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. It is possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities
additional information
-
The N-terminal region contains a sequence homologous to the chloramphenicol acetyltransferase of Bacillus pumUus, and the C-terminal region contains a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. It is possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities
-
additional information
in combination with selection for resistance to the aminoglycoside tobramycin, the aac(6')-Ie/aph(2'')-Ia gene represents an efficient marker for plastid transformation in that it produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA. No spontaneous antibiotic resistance mutants appear under tobramycin selection
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) bynickel affinity chromatography, ultrafiltration, and gel filtration
-
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by kanamycin and gentamicin affinity chromatography
-
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene aacA-aphD, synthesis and codon optimization of the gene from Staphylococcus aureus for expression in Nicotiana tabacum from the plastid genome, driven by the psbA gene promoter (PpsbA) from Chlamydomonas reinhardtii, generation of transplastomic lines with the three different aac6-aph2 vectors, recombinant expression in tobacco chloroplasts
gene aphA, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
-
gene aphSR2, sequence comparisons, recombinant expression in Escherichia coli strain BL21(DE3), coexpression of aphSR2 gene and genes pkSR1 and pkSR2, encoding serine-threonine protein kinases and localized in one cluster of the Streptomyces rimosus strain ATCC 10970 genome, causing a 2fold increase in resistance to neomycin
-
recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3). The catalytic activity of APH(2'')-IVa is independent of the presence of the 6His-tag
-
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
-
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
molecular biology
there is a need to develop alternative markers for plastid transformation to (a) extend the species range of the technology, and (b) facilitate the multistep engineering of plastid genomes, for example, by sequential introduction of multiple transgenes (supertransformation). Bifunctional aminoglycoside acetyltransferase/phosphotransferase conferring tobramycin resistance provides an efficient selectable marker for stable plastid transformation
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Toth, M.; Frase, H.; Antunes, N.T.; Vakulenko, S.B.
Novel aminoglycoside 2-phosphotransferase identified in a gram-negative pathogen
Antimicrob. Agents Chemother.
57
452-457
2013
Campylobacter jejuni (Q4VR94), Campylobacter jejuni
brenda
Shi, K.; Houston, D.R.; Berghuis, A.M.
Crystal structures of antibiotic-bound complexes of aminoglycoside 2-phosphotransferase IVa highlight the diversity in substrate binding modes among aminoglycoside kinases
Biochemistry
50
6237-6244
2011
Enterococcus casseliflavus (O68183)
brenda
Ferretti, J.; Gilmore, K.; Courvalin, P.
Nucleotide sequence analysis of the gene specifying the bifunctional 6-aminoglycoside acetyltransferase 2-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities
J. Bacteriol.
167
631-638
1986
Enterococcus faecalis (P0A0C2), Enterococcus faecalis ATCC 700802 (P0A0C2)
brenda
Badarau, A.; Shi, Q.; Chow, J.W.; Zajicek, J.; Mobashery, S.; Vakulenko, S.
Aminoglycoside 2''-phosphotransferase type IIIa from Enterococcus
J. Biol. Chem.
283
7638-7647
2008
Enterococcus sp.
brenda
Smith, C.A.; Toth, M.; Frase, H.; Byrnes, L.J.; Vakulenko, S.B.
Aminoglycoside 2-phosphotransferase IIIa (APH(2)-IIIa) prefers GTP over ATP: structural templates for nucleotide recognition in the bacterial aminoglycoside-2 kinases
J. Biol. Chem.
287
12893-12903
2012
Enterococcus gallinarum (P96762)
brenda
Shi, K.; Berghuis, A.M.
Structural basis for dual nucleotide selectivity of aminoglycoside 2-phosphotransferase IVa provides insight on determinants of nucleotide specificity of aminoglycoside kinases
J. Biol. Chem.
287
13094-13102
2012
Enterococcus casseliflavus (O68183)
brenda
Frase, H.; Toth, M.; Vakulenko, S.
Revisiting the nucleotide and aminoglycoside substrate specificity of the bifunctional aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2'')-Ia enzyme
J. Biol. Chem.
287
43262-43269
2012
Staphylococcus aureus (P0A0C1)
brenda
Tabatabaei, I.; Ruf, S.; Bock, R.
A bifunctional aminoglycoside acetyltransferase/phosphotransferase conferring tobramycin resistance provides an efficient selectable marker for plastid transformation
Plant Mol. Biol.
93
269-281
2017
Staphylococcus aureus (P0A0C1)
brenda
Smith, C.; Toth, M.; Stewart, N.; Maltz, L.; Vakulenko, S.
Structural basis for the diversity of the mechanism of nucleotide hydrolysis by the aminoglycoside-2''-phosphotransferases
Acta Crystallogr. Sect. D
75
1129-1137
2019
Enterococcus sp.
brenda
Caldwell, S.J.; Berghuis, A.M.
Plasticity of Aminoglycoside Binding to Antibiotic Kinase APH(2'')-Ia
Antimicrob. Agents Chemother.
62
e00202-18
2018
Enterococcus casseliflavus
brenda
Kaplan, E.; Guichou, J.F.; Chaloin, L.; Kunzelmann, S.; Leban, N.; Serpersu, E.H.; Lionne, C.
Aminoglycoside binding and catalysis specificity of aminoglycoside 2''-phosphotransferase IVa A thermodynamic, structural and kinetic study
Biochim. Biophys. Acta
1860
802-813
2016
Enterococcus casseliflavus
brenda
Leban, N.; Kaplan, E.; Chaloin, L.; Godreuil, S.; Lionne, C.
Kinetic characterization and molecular docking of novel allosteric inhibitors of aminoglycoside phosphotransferases
Biochim. Biophys. Acta
1861
3464-3473
2017
Enterococcus casseliflavus
brenda
Zhu, L.; Liu, R.; Liu, T.; Zou, X.; Xu, Z.; Guan, H.
A novel strategy to screen inhibitors of multiple aminoglycoside-modifying enzymes with ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry
J. Pharm. Biomed. Anal.
164
520-527
2019
Staphylococcus aureus (P0A0C1)
brenda
Rudakova, N.; Alekseeva, M.; Zakharevich, N.; Mavletova, D.; Danilenko, V.
Aminoglycoside phosphotransferase AphSR2 from Streptomyces rimosus ATCC 10970 dependence of antibiotic Resistance on serine-threonine protein kinases PkSR1 and PkSR2
Russ. J. Genet.
56
112-117
2020
Streptomyces rimosus, Streptomyces rimosus ATCC 10970
-
brenda
Caldwell, S.J.; Huang, Y.; Berghuis, A.M.
Antibiotic binding drives catalytic activation of aminoglycoside kinase APH(2'')-Ia
Structure
24
935-945
2016
Staphylococcus aureus (P0A0C1), Staphylococcus aureus
brenda
Select items on the left to see more content.
EXTERNAL LINKS (specific for EC-Number 2.7.1.190)
html completed
Use of this online version of BRENDA is free under the CC BY 4.0 license. See terms of use for full details.
Release 2023.1 (January 2023)
About BRENDA
Social media
Help
Survey
Download
Contribution
Legal
Curated at
Member of
