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Information on EC 2.7.1.190 - aminoglycoside 2''-phosphotransferase and Organism(s) Staphylococcus aureus and UniProt Accession P0A0C1
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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.
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Word Map
- 2.7.1.190
-
aph3\'-iiia
- kanamycin
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enterococci
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aac6\'-ie-aph2\'\'-ia
- faecium
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aminoglycoside-modifying
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phosphotransferases
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aph2\'\'-ic
- amikacin
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gentamicin-resistant
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aac6\'-aph2
- tobramycin
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4,6-disubstituted
- casseliflavus
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aac6'-ie
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ant6\'-ia
- gallinarum
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aminoglycoside-resistance
- lividomycin
- butirosin
-
o-phosphorylation
- sisomicin
- durans
- molecular biology
The taxonomic range for the selected organisms is: Staphylococcus aureus
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
aph(2'')-id, aminoglycoside kinase, aph(2''), aph(2'')-ia, aph(2'')-iva, aac6-aph2, aph(2'')-iiia, aminoglycoside 2''-phosphotransferase, gentamicin phosphotransferase, aminoglycoside 2''-phosphotransferase iva, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
bifunctional aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2'')-Ia
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bifunctional aminoglycoside acetyltransferase/phosphotransferase
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aminoglycoside (2'') kinase
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APH(2'')
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-
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aphD
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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.
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
GTP + kanamycin A
GDP + kanamycin A 2''-phosphate
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-
-
?
additional information
?
-
GTP is the exclusive phosphate donor at intracellular nucleotide levels
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-
?
additional information
?
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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'')
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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
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-
?
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
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additional information
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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
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additional information
GTP is more qualified as a phosphate donor than ATP to cause phosphorylation events of AAC(6')-APH(2'')
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Mg2+
required, magnesium ions, Mg1 and Mg2, are consistently resolved with coordinating water molecules
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
Michaelis-Menten kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
additional information
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
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
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
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
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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
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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
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
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
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)
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)
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)
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Release 2023.1 (January 2023)
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