2.7.1.190: aminoglycoside 2''-phosphotransferase
This is an abbreviated version!
For detailed information about aminoglycoside 2''-phosphotransferase, go to the full flat file.
Word Map on EC 2.7.1.190
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2.7.1.190
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aph3\'-iiia
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kanamycin
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enterococci
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aac6\'-ie-aph2\'\'-ia
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faecium
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aminoglycoside-modifying
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phosphotransferases
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aph2\'\'-ic
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amikacin
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gentamicin-resistant
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aac6\'-aph2
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tobramycin
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4,6-disubstituted
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casseliflavus
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aac6'-ie
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ant6\'-ia
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gallinarum
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aminoglycoside-resistance
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lividomycin
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butirosin
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o-phosphorylation
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sisomicin
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durans
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molecular biology
- 2.7.1.190
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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
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o-phosphorylation
- sisomicin
- durans
- molecular biology
Reaction
Synonyms
AAC(6')-APH(2''), AAC(6')-Ie/APH(2'')-Ia, aac6-aph2, aacA-aphD, AME, aminoglycoside (2'') kinase, aminoglycoside 2''-phosphotransferase IVa, aminoglycoside 2''-phosphotransferase type IIIa, aminoglycoside kinase, aminoglycoside phosphotransferase, aminoglycoside-2''-phosphotransferase, aminoglycoside-2''-phosphotransferase type IVa, aminoglycoside-modification enzyme, APH, APH(2''), APH(2'')-Ia, APH(2'')-Ia aminoglycoside resistance enzyme, APH(2'')-Id, Aph(2'')-If, APH(2'')-IIIa, 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, gentamicin phosphotransferase, gentamicin resistance protein
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General Information on EC 2.7.1.190 - aminoglycoside 2''-phosphotransferase
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GENERAL INFORMATION 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
evolution
malfunction
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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
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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
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Streptomyces rimosus ATCC 10970 contains 14 genes annotated as aminoglycoside phosphotransferases in its genome: aphSR1-aphSR14
evolution
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Streptomyces rimosus ATCC 10970 contains 14 genes annotated as aminoglycoside phosphotransferases in its genome: aphSR1-aphSR14
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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
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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
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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
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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
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additional information
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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
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coexpression of aphSR2 gene and genes pkSR1 and pkSR2, encoding serine-threonine protein kinases, causes a 2fold increase in resistance to neomycin
additional information
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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
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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
additional information
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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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