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IUBMB CommentsRequires 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.
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
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ATP + gentamicin
ADP + gentamicin 2''-phosphate
-
GTP is preferred over ATP
-
?
ATP + kanamycin
ADP + kanamycin 2''-phosphate
ATP + kanamycin A
ADP + kanamycin A 2''-phosphate
-
-
-
-
?
ATP + tobramycin
ADP + tobramycin 2''-phosphate
GTP + amikacin
GDP + amikacin 2''-phosphate
GTP + arbekacin
GDP + arbekacin 2''-phosphate
GTP + dibekacin
GDP + dibekacin 2''-phosphate
GTP + G418
GDP + G418 2''-phosphate
-
-
-
-
?
GTP + gentamicin
GDP + gentamicin 2''-phosphate
GTP + gentamicin C
GDP + gentamicin C 2''-phosphate
GTP + gentamicin C1
GDP + gentamicin C1 2''-phosphate
-
-
-
?
GTP + hygromycin
GDP + hygromycin 2''-phosphate
GTP + isepamicin
GDP + isepamicin 2''-phosphate
-
-
-
?
GTP + isepamycin
GDP + isepamycin 2''-phosphate
-
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
GTP + kanamycin A
GDP + kanamycin A 2''-phosphate
GTP + kanamycin B
GDP + kanamycin B 2''-phosphate
GTP + lividomycin
GDP + lividomycin 2''-phosphate
GTP + neomycin
GDP + neomycin 2''-phosphate
GTP + neomycin B
GDP + neomycin B 2''-phosphate
-
-
-
?
GTP + netilmicin
GDP + netilmicin 2''-phosphate
GTP + netilmycin
GDP + netilmycin 2''-phosphate
-
-
-
-
?
GTP + ribostamycin
GDP + ribostamycin 2''-phosphate
GTP + sisomicin
GDP + sisomicin 2''-phosphate
GTP + sisomycin
GDP + sisomycin 2''-phosphate
-
-
-
-
?
GTP + streptomycin
GDP + streptomycin 2''-phosphate
-
-
-
-
?
GTP + tobramycin
GDP + tobramycin 2''-phosphate
ITP + dibekacin
IDP + dibekacin 2''-phosphate
-
-
-
-
?
UTP + tobramycin
UDP + tobramycin 2''-phosphate
-
-
-
-
?
additional information
?
-
ATP + kanamycin
ADP + kanamycin 2''-phosphate
-
-
-
-
?
ATP + kanamycin
ADP + kanamycin 2''-phosphate
-
-
-
?
ATP + tobramycin
ADP + tobramycin 2''-phosphate
-
-
-
-
?
ATP + tobramycin
ADP + tobramycin 2''-phosphate
-
-
-
?
GTP + amikacin
GDP + amikacin 2''-phosphate
-
-
-
?
GTP + amikacin
GDP + amikacin 2''-phosphate
-
-
-
?
GTP + amikacin
GDP + amikacin 2''-phosphate
-
-
-
-
?
GTP + amikacin
GDP + amikacin 2''-phosphate
-
-
-
-
?
GTP + arbekacin
GDP + arbekacin 2''-phosphate
-
-
-
?
GTP + arbekacin
GDP + arbekacin 2''-phosphate
-
-
-
?
GTP + dibekacin
GDP + dibekacin 2''-phosphate
-
-
-
?
GTP + dibekacin
GDP + dibekacin 2''-phosphate
-
-
-
-
?
GTP + dibekacin
GDP + dibekacin 2''-phosphate
-
-
-
?
GTP + gentamicin
GDP + gentamicin 2''-phosphate
-
-
-
-
?
GTP + gentamicin
GDP + gentamicin 2''-phosphate
-
-
-
?
GTP + gentamicin
GDP + gentamicin 2''-phosphate
-
-
-
-
?
GTP + gentamicin
GDP + gentamicin 2''-phosphate
-
-
-
-
?
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 + gentamicin C
GDP + gentamicin C 2''-phosphate
-
-
-
?
GTP + hygromycin
GDP + hygromycin 2''-phosphate
-
-
-
-
?
GTP + hygromycin
GDP + hygromycin 2''-phosphate
-
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
-
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
-
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
-
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
-
-
-
-
?
GTP + kanamycin A
GDP + kanamycin A 2''-phosphate
-
-
-
?
GTP + kanamycin A
GDP + kanamycin A 2''-phosphate
-
-
-
?
GTP + kanamycin A
GDP + kanamycin A 2''-phosphate
-
-
-
-
?
GTP + kanamycin B
GDP + kanamycin B 2''-phosphate
-
-
-
?
GTP + kanamycin B
GDP + kanamycin B 2''-phosphate
-
-
-
?
GTP + lividomycin
GDP + lividomycin 2''-phosphate
-
-
-
-
?
GTP + lividomycin
GDP + lividomycin 2''-phosphate
-
enzyme binding structure analysis, overview
-
-
?
GTP + neomycin
GDP + neomycin 2''-phosphate
-
-
-
-
?
GTP + neomycin
GDP + neomycin 2''-phosphate
-
-
-
-
?
GTP + netilmicin
GDP + netilmicin 2''-phosphate
-
-
-
?
GTP + netilmicin
GDP + netilmicin 2''-phosphate
-
-
-
-
?
GTP + netilmicin
GDP + netilmicin 2''-phosphate
-
-
-
?
GTP + ribostamycin
GDP + ribostamycin 2''-phosphate
-
-
-
-
?
GTP + ribostamycin
GDP + ribostamycin 2''-phosphate
-
-
-
?
GTP + sisomicin
GDP + sisomicin 2''-phosphate
-
-
-
?
GTP + sisomicin
GDP + sisomicin 2''-phosphate
-
-
-
-
?
GTP + sisomicin
GDP + sisomicin 2''-phosphate
-
-
-
?
GTP + tobramycin
GDP + tobramycin 2''-phosphate
-
-
-
?
GTP + tobramycin
GDP + tobramycin 2''-phosphate
-
-
-
?
GTP + tobramycin
GDP + tobramycin 2''-phosphate
-
-
-
?
GTP + tobramycin
GDP + tobramycin 2''-phosphate
-
-
-
-
?
GTP + tobramycin
GDP + tobramycin 2''-phosphate
-
-
-
-
?
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
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-
-
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'')
-
-
-
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ATP + kanamycin
ADP + kanamycin 2''-phosphate
-
-
-
-
?
ATP + tobramycin
ADP + tobramycin 2''-phosphate
-
-
-
-
?
GTP + amikacin
GDP + amikacin 2''-phosphate
GTP + dibekacin
GDP + dibekacin 2''-phosphate
-
-
-
-
?
GTP + gentamicin
GDP + gentamicin 2''-phosphate
GTP + gentamicin C1
GDP + gentamicin C1 2''-phosphate
-
-
-
?
GTP + hygromycin
GDP + hygromycin 2''-phosphate
GTP + isepamycin
GDP + isepamycin 2''-phosphate
-
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
GTP + kanamycin A
GDP + kanamycin A 2''-phosphate
-
-
-
?
GTP + lividomycin
GDP + lividomycin 2''-phosphate
-
-
-
-
?
GTP + neomycin
GDP + neomycin 2''-phosphate
GTP + neomycin B
GDP + neomycin B 2''-phosphate
-
-
-
?
GTP + netilmycin
GDP + netilmycin 2''-phosphate
-
-
-
-
?
GTP + ribostamycin
GDP + ribostamycin 2''-phosphate
GTP + sisomycin
GDP + sisomycin 2''-phosphate
-
-
-
-
?
GTP + streptomycin
GDP + streptomycin 2''-phosphate
-
-
-
-
?
GTP + tobramycin
GDP + tobramycin 2''-phosphate
additional information
?
-
GTP is the exclusive phosphate donor at intracellular nucleotide levels
-
-
?
GTP + amikacin
GDP + amikacin 2''-phosphate
-
-
-
-
?
GTP + amikacin
GDP + amikacin 2''-phosphate
-
-
-
-
?
GTP + gentamicin
GDP + gentamicin 2''-phosphate
-
-
-
-
?
GTP + gentamicin
GDP + gentamicin 2''-phosphate
-
-
-
?
GTP + gentamicin
GDP + gentamicin 2''-phosphate
-
-
-
-
?
GTP + gentamicin
GDP + gentamicin 2''-phosphate
-
-
-
-
?
GTP + hygromycin
GDP + hygromycin 2''-phosphate
-
-
-
-
?
GTP + hygromycin
GDP + hygromycin 2''-phosphate
-
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
-
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
-
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
-
-
-
-
?
GTP + kanamycin
GDP + kanamycin 2''-phosphate
-
-
-
-
?
GTP + neomycin
GDP + neomycin 2''-phosphate
-
-
-
-
?
GTP + neomycin
GDP + neomycin 2''-phosphate
-
-
-
-
?
GTP + ribostamycin
GDP + ribostamycin 2''-phosphate
-
-
-
-
?
GTP + ribostamycin
GDP + ribostamycin 2''-phosphate
-
-
-
?
GTP + tobramycin
GDP + tobramycin 2''-phosphate
-
-
-
-
?
GTP + tobramycin
GDP + tobramycin 2''-phosphate
-
-
-
-
?
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0.0008 - 0.0062
arbekacin
0.0003 - 0.0012
dibekacin
0.0007 - 0.006
gentamicin C
0.56
isepamicin
pH 7.0, 22°C
0.039
ITP
-
pH 7.5, 25°C, cosubstrate kanamycin A
0.00441
kanamycin
pH 7.5, 37°C
0.0011 - 0.0045
kanamycin A
0.00055
kanamycin B
pH 7.0, 22°C
0.0008 - 0.0016
sisomicin
0.001
tobramycin
above 0.001, pH 7.0, 22°C
0.1
UTP
-
pH 7.5, 25°C, cosubstrate kanamycin A
additional information
additional information
-
0.033
amikacin
pH 7.0, 22°C
0.26
amikacin
pH 7.0, 22°C
0.0008
arbekacin
pH 7.0, 22°C
0.0062
arbekacin
pH 7.0, 22°C
0.055
ATP
mutant F95M, pH 7.5, 37°C
0.069
ATP
wild-type, pH 7.5, 37°C
0.072
ATP
mutant F95Y, pH 7.5, 37°C
0.09
ATP
-
pH 7.5, 25°C, cosubstrate kanamycin A
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
0.0003
dibekacin
pH 7.0, 22°C
0.0012
dibekacin
pH 7.0, 22°C
0.0007
gentamicin C
pH 7.0, 22°C
0.006
gentamicin C
pH 7.0, 22°C
0.0008
GTP
pH 7.0, 22°C
0.0045
GTP
-
pH 7.5, 25°C, cosubstrate kanamycin A
0.04
GTP
wild-type, pH not specified in the publication, temperature not specified in the publication
0.049
GTP
mutant F95Y, pH 7.5, 37°C
0.094
GTP
mutant Y92A, pH not specified in the publication, temperature not specified in the publication
0.156
GTP
mutant F95M, pH 7.5, 37°C
0.168
GTP
wild-type, pH 7.5, 37°C
0.0011
kanamycin A
pH 7.0, 22°C
0.0045
kanamycin A
-
pH 7.5, 25°C
0.0041
netilmicin
pH 7.0, 22°C
0.01
netilmicin
pH 7.0, 22°C
0.0008
sisomicin
pH 7.0, 22°C
0.0016
sisomicin
pH 7.0, 22°C
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
-
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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
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
-
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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
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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
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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)
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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
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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
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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
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site-directed mutagenesis, the point mutant and wild-type enzymes have the same structure
S214A
site-directed mutagenesis
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
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
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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
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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
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Enterococcus casseliflavus (O68183)
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Staphylococcus aureus (P0A0C1)
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Staphylococcus aureus (P0A0C1)
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Enterococcus sp.
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Plasticity of Aminoglycoside Binding to Antibiotic Kinase APH(2'')-Ia
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Enterococcus casseliflavus
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Enterococcus casseliflavus
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Kinetic characterization and molecular docking of novel allosteric inhibitors of aminoglycoside phosphotransferases
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3464-3473
2017
Enterococcus casseliflavus
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Staphylococcus aureus (P0A0C1)
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Streptomyces rimosus, Streptomyces rimosus ATCC 10970
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Structure
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2016
Staphylococcus aureus (P0A0C1), Staphylococcus aureus
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