Information on EC 1.14.13.231 - tetracycline 11a-monooxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.13.231
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RECOMMENDED NAME
GeneOntology No.
tetracycline 11a-monooxygenase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
RH + [reduced NADPH-hemoprotein reductase] + O2 = ROH + [oxidized NADPH-hemoprotein reductase] + H2O
show the reaction diagram
tetracycline + NADPH + H+ + O2 = 11a-hydroxytetracycline + NADP+ + H2O
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
tetracycline resistance
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Tetracycline biosynthesis
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Biosynthesis of antibiotics
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SYSTEMATIC NAME
IUBMB Comments
tetracycline,NADPH:oxygen oxidoreductase (11a-hydroxylating)
A flavoprotein (FAD). This bacterial enzyme confers resistance to all clinically relevant tetracyclines when expressed under aerobic conditions. The hydroxylated products are very unstable and lead to intramolecular cyclization and non-enzymic breakdown to undefined products.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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tigecycline is a substrate for TetX and bacterial strains containing the TetX gene are resistant to tigecycline due to the modification of tigecycline by TetX to form 11a-hydroxytigecycline, which has a weakened ability to inhibit protein translation compared with tigecycline. 11a-Hydroxytigecycline forms a weaker complex with magnesium than tigecycline, magnesium coordination is critical for binding tetracycline to the ribosome
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
anhydrotetracycline + NADPH + H+ + O2
11a-hydroxyanhydrotetracycline + NADP+ + H2O
show the reaction diagram
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-
-
?
chlorotetracycline + NADPH + H+ + O2
11a-hydroxychlorotetracycline + NADP+ + H2O
show the reaction diagram
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-
-
-
?
chlortetracycline + NADPH + H+ + O2
11a-hydroxychlortetracycline + NADP+ + H2O
show the reaction diagram
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-
-
?
chlortetracycline + reduced flavoprotein + O2
11a-hydroxy-chlortetracycline + oxidized flavoprotein + H2O
show the reaction diagram
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-
-
-
?
demeclocycline + NADPH + H+ + O2
11a-hydroxydemeclocycline + NADP+ + H2O
show the reaction diagram
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-
-
?
demeclocycline + reduced flavoprotein + O2
11a-hydroxy-demeclocycline + oxidized flavoprotein + H2O
show the reaction diagram
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-
-
?
doxycycline + NADPH + H+ + O2
11a-hydroxydoxycycline + NADP+ + H2O
show the reaction diagram
doxycycline + reduced flavoprotein + O2
11a-hydroxy-doxycycline + oxidized flavoprotein + H2O
show the reaction diagram
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-
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?
minocycline + NADPH + H+ + O2
11a-hydroxyminocycline + NADP+ + H2O
show the reaction diagram
minocycline + reduced flavoprotein + O2
11a-hydroxy-minocycline + oxidized flavoprotein + H2O
show the reaction diagram
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-
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-
?
oxytetracycline + reduced flavoprotein + NADPH + O2
11a-hydroxy-oxytetracycline + oxidized flavoprotein + H2O + NADP+
show the reaction diagram
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?
tetracycline + NADPH + H+ + O2
11a-hydroxytetracycline + NADP+ + H2O
show the reaction diagram
tetracycline + reduced flavoprotein + O2
11a-hydroxy-tetracycline + oxidized flavoprotein + H2O
show the reaction diagram
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?
tigecycline + NADPH + H+ + O2
11a-hydroxytigecycline + NADP+ + H2O
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
chlortetracycline + NADPH + H+ + O2
11a-hydroxychlortetracycline + NADP+ + H2O
show the reaction diagram
Q93L51
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-
-
?
demeclocycline + NADPH + H+ + O2
11a-hydroxydemeclocycline + NADP+ + H2O
show the reaction diagram
Q93L51
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?
doxycycline + NADPH + H+ + O2
11a-hydroxydoxycycline + NADP+ + H2O
show the reaction diagram
Q93L51
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-
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?
minocycline + NADPH + H+ + O2
11a-hydroxyminocycline + NADP+ + H2O
show the reaction diagram
Q93L51
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?
tetracycline + NADPH + H+ + O2
11a-hydroxytetracycline + NADP+ + H2O
show the reaction diagram
Q93L51
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?
additional information
?
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Q93L51
the enzyme shows a broad tetracycline antibiotic spectrum and a requirement for molecular oxygen and NADPH in antibiotic degradation. The tetracycline products are unstable at neutral pH, but mass spectral and NMR characterization under acidic conditions support initial monohydroxylation at position 11a followed by intramolecular cyclization and non-enzymatic breakdown to other undefined products
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.076
alpha-doxycycline
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pH 8.5, temperature not specified in the publication
0.13 - 0.16
anhydrotetracycline
0.11
chlorotetracycline
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presence of 0.0002% arabinose, pH not specified in the publication, temperature not specified in the publication
0.11
chlortetracycline
0.0199 - 0.02
demeclocycline
0.083 - 0.084
doxycycline
0.028 - 0.0284
minocycline
0.133
NADPH
0.076
oxytetracycline
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pH 8.5
0.054
tetracycline
0.044
tigecycline
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pH 8.5, temperature not specified in the publication
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.3
alpha-doxycycline
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pH 8.5, temperature not specified in the publication
0.4 - 0.7
anhydrotetracycline
0.3
chlorotetracycline
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presence of 0.0002% arabinose, pH not specified in the publication, temperature not specified in the publication
0.3
chlortetracycline
0.2
demeclocycline
0.6 - 0.63
doxycycline
0.12
minocycline
1.11
NADPH
1.3
oxytetracycline
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pH 8.5
0.32
tetracycline
0.36
tigecycline
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pH 8.5, temperature not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
17
alpha-doxycycline
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pH 8.5, temperature not specified in the publication
2.7
chlortetracycline
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pH 8.5, temperature not specified in the publication
10
demeclocycline
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pH 8.5, temperature not specified in the publication
7.5
doxycycline
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pH 8.5, temperature not specified in the publication
4.1
minocycline
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pH 8.5, temperature not specified in the publication
8.3
NADPH
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pH 8.5, temperature not specified in the publication
5.9
tetracycline
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pH 8.5, temperature not specified in the publication
8.3
tigecycline
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pH 8.5, temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
43700
x * 43700, calculated
44000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 43700, calculated
monomer
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x * 44000, SDS-PAGE and deduced from gene sequence
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with tetracyclines minocycline and tigecycline at 2.18 and 2.30 A resolution, respectively. Both tetracyclines bind in a large tunnel-shaped active site in close contact to the cofactor FAD, preoriented for regioselective hydroxylation to 11alpha-hydroxytetracyclines. The bulky 9-tert-butylglycylamido substituent of tigecycline is solvent-exposed and does not interfere with TetX binding. In the TetX-minocycline complex a second binding site for a minocycline dimer is observed close to the active-site entrance. The putative dioxygen-binding cavities are located in the substrate-binding domain next to the active site
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structure at 2.8 A resolution and comparison with that of the weakly homologous Pseudomonas fluorescens parahydroxybenzoate hydroxylase
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structure determinations at 2.1 A resolution of native TetX and its complexes with tetracyclines. Domain 1 exhibits the Rossmann fold responsible for binding of the coenzyme FAD through its adenosine monophosphate component, which is linked to the flavin mononucleotide containing the catalytically active isoalloxazine moiety. The second domain with an extended 7-stranded beta-sheet is positioned like a shield on top of the flavin-binding domain covered by five alpha-helices and is responsible for substrate recognition. A long C-terminal alpha-helix stabilizes the association of the two domains
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant protein, purified protein gives a yellow solution
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D311A
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mutation in a highly conserved residue in the FAD-binding site, inactive
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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construction of a sensitive fluorescent reporter system to detect and characterize the activity of enzymes that act upon the antibiotic, tetracycline and its derivatives. In this system, expression of the lux operon is regulated by the tetracycline repressor, TetR, which is expressed from the same plasmid under the control of an arabinose-inducible promoter. Addition of very low concentrations of tetracycline derivatives, well below growth inhibitory concentrations, result in luminescence production. Introduction of a plasmid expressing TetX, a tetracycline-inactivating enzyme, causes a marked loss in luminescence due to enzyme-mediated reduction in the intracellular tetracycline concentration
biotechnology
degradation