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Information on EC 1.14.13.231 - tetracycline 11a-monooxygenase
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1.14.13.231 tetracycline 11a-monooxygenaseIUBMB Comments
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.
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The enzyme appears in viruses and cellular organisms
Reaction Schemes
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Synonyms
tetx2, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
BN1088_100004
tetracylcine resistance protein TetX
TetX
TetX
-
-
gene name
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
RH + [reduced NADPH-hemoprotein reductase] + O2 = ROH + [oxidized NADPH-hemoprotein reductase] + H2O
tetracycline + NADPH + H+ + O2 = 11a-hydroxytetracycline + NADP+ + H2O
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RH + [reduced NADPH-hemoprotein reductase] + O2 = ROH + [oxidized NADPH-hemoprotein reductase] + H2O
tetracycline substrates, initial monohydroxylation at position 11a followed by intramolecular cyclization and non-enzymatic breakdown to undefined products
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RH + [reduced NADPH-hemoprotein reductase] + O2 = ROH + [oxidized NADPH-hemoprotein reductase] + H2O
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-
-
-
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PATHWAY SOURCE
PATHWAYS
MetaCyc
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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.
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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
anhydrotetracycline + NADPH + H+ + O2
11a-hydroxyanhydrotetracycline + NADP+ + H2O
-
-
-
-
?
chlorotetracycline + NADPH + H+ + O2
11a-hydroxychlorotetracycline + NADP+ + H2O
-
-
-
-
?
chlortetracycline + NADPH + H+ + O2
11a-hydroxychlortetracycline + NADP+ + H2O
-
-
-
?
chlortetracycline + reduced flavoprotein + O2
11a-hydroxy-chlortetracycline + oxidized flavoprotein + H2O
-
-
-
-
?
demeclocycline + NADPH + H+ + O2
11a-hydroxydemeclocycline + NADP+ + H2O
-
-
-
?
demeclocycline + reduced flavoprotein + O2
11a-hydroxy-demeclocycline + oxidized flavoprotein + H2O
-
-
-
-
?
doxycycline + reduced flavoprotein + O2
11a-hydroxy-doxycycline + oxidized flavoprotein + H2O
-
-
-
-
?
minocycline + reduced flavoprotein + O2
11a-hydroxy-minocycline + oxidized flavoprotein + H2O
-
-
-
-
?
oxytetracycline + reduced flavoprotein + NADPH + O2
11a-hydroxy-oxytetracycline + oxidized flavoprotein + H2O + NADP+
-
-
-
-
?
tetracycline + reduced flavoprotein + O2
11a-hydroxy-tetracycline + oxidized flavoprotein + H2O
-
-
-
-
?
doxycycline + NADPH + H+ + O2
11a-hydroxydoxycycline + NADP+ + H2O
-
-
-
-
?
doxycycline + NADPH + H+ + O2
11a-hydroxydoxycycline + NADP+ + H2O
-
-
-
?
minocycline + NADPH + H+ + O2
11a-hydroxyminocycline + NADP+ + H2O
-
-
-
?
minocycline + NADPH + H+ + O2
11a-hydroxyminocycline + NADP+ + H2O
-
-
-
-
?
tetracycline + NADPH + H+ + O2
11a-hydroxytetracycline + NADP+ + H2O
-
-
-
?
tetracycline + NADPH + H+ + O2
11a-hydroxytetracycline + NADP+ + H2O
-
-
-
?
tigecycline + NADPH + H+ + O2
11a-hydroxytigecycline + NADP+ + H2O
-
-
-
-
?
tigecycline + NADPH + H+ + O2
11a-hydroxytigecycline + NADP+ + H2O
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-
-
-
?
additional information
?
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tetracycline substrates, initial monohydroxylation at position 11a followed by intramolecular cyclization and non-enzymatic breakdown to undefined products
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additional information
?
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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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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
chlortetracycline + NADPH + H+ + O2
11a-hydroxychlortetracycline + NADP+ + H2O
Q93L51
-
-
-
?
demeclocycline + NADPH + H+ + O2
11a-hydroxydemeclocycline + NADP+ + H2O
Q93L51
-
-
-
?
doxycycline + NADPH + H+ + O2
11a-hydroxydoxycycline + NADP+ + H2O
Q93L51
-
-
-
?
minocycline + NADPH + H+ + O2
11a-hydroxyminocycline + NADP+ + H2O
Q93L51
-
-
-
?
tetracycline + NADPH + H+ + O2
11a-hydroxytetracycline + NADP+ + H2O
Q93L51
-
-
-
?
additional information
?
-
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
LITERATURE
IMAGE
FAD
-
FAD is bound non-covalently in an elongated, so-called IN conformation with the adenine and isoalloxazine moieties distal to each other
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.076
alpha-doxycycline
-
pH 8.5, temperature not specified in the publication
0.11
chlorotetracycline
-
presence of 0.0002% arabinose, pH not specified in the publication, temperature not specified in the publication
0.044
tigecycline
-
pH 8.5, temperature not specified in the publication
0.13
anhydrotetracycline
-
presence of 0.0002% arabinose, pH not specified in the publication, temperature not specified in the publication
0.16
anhydrotetracycline
-
presence of 0.02% arabinose, pH not specified in the publication, temperature not specified in the publication
0.11
chlortetracycline
-
pH 8.5, temperature not specified in the publication
0.02
demeclocycline
-
pH 8.5, temperature not specified in the publication
0.083
doxycycline
-
presence of 0.0002% arabinose, pH not specified in the publication, temperature not specified in the publication
0.084
doxycycline
-
pH 8.5, temperature not specified in the publication
0.028
minocycline
-
pH 8.5, temperature not specified in the publication
0.133
NADPH
-
pH 8.5, temperature not specified in the publication
0.054
tetracycline
-
pH 8.5, temperature not specified in the publication
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.3
alpha-doxycycline
-
pH 8.5, temperature not specified in the publication
0.3
chlorotetracycline
-
presence of 0.0002% arabinose, pH not specified in the publication, temperature not specified in the publication
0.4
anhydrotetracycline
-
presence of 0.02% arabinose, pH not specified in the publication, temperature not specified in the publication
0.7
anhydrotetracycline
-
presence of 0.0002% arabinose, pH not specified in the publication, temperature not specified in the publication
0.3
chlortetracycline
-
pH 8.5, temperature not specified in the publication
0.2
demeclocycline
-
pH 8.5, temperature not specified in the publication
0.6
doxycycline
-
presence of 0.0002% arabinose, pH not specified in the publication, temperature not specified in the publication
0.63
doxycycline
-
pH 8.5, temperature not specified in the publication
0.12
minocycline
-
pH 8.5, temperature not specified in the publication
1.11
NADPH
-
pH 8.5, temperature not specified in the publication
0.32
tetracycline
-
pH 8.5, temperature not specified in the publication
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
17
alpha-doxycycline
-
pH 8.5, temperature not specified in the publication
2.7
chlortetracycline
-
pH 8.5, temperature not specified in the publication
10
demeclocycline
-
pH 8.5, temperature not specified in the publication
7.5
doxycycline
-
pH 8.5, temperature not specified in the publication
4.1
minocycline
-
pH 8.5, temperature not specified in the publication
8.3
NADPH
-
pH 8.5, temperature not specified in the publication
5.9
tetracycline
-
pH 8.5, temperature not specified in the publication
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
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
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
Sequence
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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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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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D311A
-
mutation in a highly conserved residue in the FAD-binding site, inactive
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PURIFICATION/commentary
ORGANISM
UNIPROT
LITERATURE
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CLONED/commentary
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
-
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
biotechnology
I6YQD0
protocol for the nuclear transformation of Chlamydomonas reinhardtii using tetX as a selectable marker that confers stable resistance to tetracycline up to 100 microg/ml. TetX may be used to transform Chlamydomonas reinhardtii chloroplasts, related microalgae and other aerobic organisms sensitive to any tetracycline antibiotic
biotechnology
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protocol for the nuclear transformation of Chlamydomonas reinhardtii using tetX as a selectable marker that confers stable resistance to tetracycline up to 100 microg/ml. TetX may be used to transform Chlamydomonas reinhardtii chloroplasts, related microalgae and other aerobic organisms sensitive to any tetracycline antibiotic
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degradation
addition of Escherichia coli overexpressing TetX to soil bacterial enrichment cultures along with varying levels of tetracycline affects community-wide tetracycline resistance levels. Soil microbial communities develop lower levels of tetracycline resistance upon exposure to 25 microg/ml of tetracycline when an Escherichia coli expressing TetX is present (6% of cultivable bacteria are resistant to 40 microg/ml tetracycline). In the absence of TetX activity, a similar tetracycline exposure selects for greater levels of resistant bacteria in the soil microbial community (90% of cultivable bacteria are resistant to 40 microg/ml tetracycline)
degradation
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addition of Escherichia coli overexpressing TetX to soil bacterial enrichment cultures along with varying levels of tetracycline affects community-wide tetracycline resistance levels. Soil microbial communities develop lower levels of tetracycline resistance upon exposure to 25 microg/ml of tetracycline when an Escherichia coli expressing TetX is present (6% of cultivable bacteria are resistant to 40 microg/ml tetracycline). In the absence of TetX activity, a similar tetracycline exposure selects for greater levels of resistant bacteria in the soil microbial community (90% of cultivable bacteria are resistant to 40 microg/ml tetracycline)
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Yang, W.; Moore, I.F.; Koteva, K.P.; Bareich, D.C.; Hughes, D.W.; Wright, G.D.
TetX is a flavin-dependent monooxygenase conferring resistance to tetracycline antibiotics
J. Biol. Chem.
279
52346-52352
2004
Bacteroides fragilis, Bacteroides thetaiotaomicron (Q93L51)
brenda
Moore, I.F.; Hughes, D.W.; Wright, G.D.
Tigecycline is modified by the flavin-dependent monooxygenase TetX
Biochemistry
44
11829-11835
2005
Bacteroides fragilis
brenda
Volkers, G.; Damas, J.M.; Palm, G.J.; Panjikar, S.; Soares, C.M.; Hinrichs, W.
Putative dioxygen-binding sites and recognition of tigecycline and minocycline in the tetracycline-degrading monooxygenase TetX
Acta Crystallogr. Sect. D
69
1758-1767
2013
Bacteroides thetaiotaomicron (Q93L51)
brenda
Yu, Z.; Reichheld, S.E.; Cuthbertson, L.; Nodwell, J.R.; Davidson, A.R.
Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system
BMC Biochem.
11
34
2010
Bacteroides fragilis
brenda
Volkers, G.; Palm, G.J.; Weiss, M.S.; Wright, G.D.; Hinrichs, W.
Structural basis for a new tetracycline resistance mechanism relying on the TetX monooxygenase
FEBS Lett.
585
1061-1066
2011
Bacteroides thetaiotaomicron (Q93L51)
brenda
Ghosh, S.; LaPara, T.M.; Sadowsky, M.J.
Transformation of tetracycline by TetX and its subsequent degradation in a heterologous host
FEMS Microbiol. Ecol.
91
fiv059
2015
Sphingobacterium sp. (A0A077XNF8), Sphingobacterium sp. PM2-P1-29 (A0A077XNF8)
brenda
Speer, B.S.; Bedzyk, L.; Salyers, A.A.
Evidence that a novel tetracycline resistance gene found on two Bacteroides transposons encodes an NADP-requiring oxidoreductase
J. Bacteriol.
173
176-183
1991
Bacteroides fragilis (Q01911)
brenda
Garcia-Echauri, S.A.; Cardineau, G.A.
TETX: a novel nuclear selection marker for Chlamydomonas reinhardtii transformation
Plant Methods
11
27
2015
Enterobacteriaceae bacterium (I6YQD0), Enterobacteriaceae bacterium SL1 (I6YQD0)
brenda
Walkiewicz, K.; Davlieva, M.; Wu, G.; Shamoo, Y.
Crystal structure of Bacteroides thetaiotaomicron TetX2: A tetracycline degrading monooxygenase at 2.8 Ã… resolution
Proteins
79
2335-2340
2011
Bacteroides thetaiotaomicron, Bacteroides thetaiotaomicron (Q93L51)
brenda
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