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Information on EC 1.13.11.24 - quercetin 2,3-dioxygenase and Organism(s) Bacillus subtilis and UniProt Accession P42106
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1.13.11.24 quercetin 2,3-dioxygenaseIUBMB Comments
The enzyme from Aspergillus sp. is a copper protein whereas that from Bacillus subtilis contains iron. Quercetin is a flavonol (5,7,3',4'-tetrahydroxyflavonol).
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The taxonomic range for the selected organisms is: Bacillus subtilis
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
pirin, quercetinase, quercetin 2,3-dioxygenase, 2,3qd, 2,3-qd, flavonol 2,4-dioxygenase, mn-qdo, quercetin dioxygenase, quercetin 2,4-dioxygenase, vdqase, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
flavonol 2,4-oxygenase
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quercetinase
quercetinase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
quercetin + O2 = 2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoate + CO + H+
structure-function analysis of the active site
quercetin + O2 = 2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoate + CO + H+
the enzyme incorporates both atoms of dioxygen into the substrate by cleaving the central heterocycle ring and releasing CO. The enzyme activates quercetin through deprotonation and the proton acceptor-Glu69 needs to reorient for the reaction to proceed. Energy profiles and reaction schemes for nonenzymatic nitroxygenation of quercetin monoanion. Transient and intermediate structures, catalytic mechaanism, detailed overview
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
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oxidation
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reduction
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PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
quercetin:oxygen 2,3-oxidoreductase (decyclizing)
The enzyme from Aspergillus sp. is a copper protein whereas that from Bacillus subtilis contains iron. Quercetin is a flavonol (5,7,3',4'-tetrahydroxyflavonol).
CAS REGISTRY NUMBER
COMMENTARY
9075-67-6
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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
fisetin + O2
2-[[(3,4-dihydroxyphenyl)carbonyl]oxy]-4-hydroxybenzoate + CO
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1.22% activity compared to quercetin
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?
galangin + O2
2,4-dihydroxy-6-[(phenylcarbonyl)oxy]benzoate + CO
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110% activity compared to quercetin
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?
tamarixetin + O2
2,4-dihydroxy-6-[[(3-hydroxy-4-methoxyphenyl)carbonyl]oxy]benzoate + CO
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82.4% activity compared to quercetin
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?
quercetin + O2
2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoate + CO + H+
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?
quercetin + O2
2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoate + CO + H+
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quercetin dioxygenase catalyzes the oxidation of the flavonol quercetin with dioxygen, cleaving the central heterocyclic ring and releasing CO
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?
quercetin + O2
2-protocatechoylphloroglucinolcarboxylate + CO
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?
quercetin + O2
2-protocatechoylphloroglucinolcarboxylate + CO
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100% activity
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?
additional information
?
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the enzyme opens up two C-C bonds of the heterocyclic ring of quercetin, a widespread plant flavonol
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?
additional information
?
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Mn-QDO in absence of O2 shows ability to react with nitroxyl (HNO)-singly reduced form of NO. HNO is incorporated into quercetin in the same manneras dioxygen, yet the reaction is strictly regioselective, as the only product is 2-((3,4-dihydroxyphenyl)(imino)methoxy)-4,6-dihydroxybenzoate
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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
?
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the enzyme opens up two C-C bonds of the heterocyclic ring of quercetin, a widespread plant flavonol
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?
quercetin + O2
2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoate + CO + H+
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?
quercetin + O2
2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoate + CO + H+
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quercetin dioxygenase catalyzes the oxidation of the flavonol quercetin with dioxygen, cleaving the central heterocyclic ring and releasing CO
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?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Iron
different coordination geometry in the two active sites of the dimer
Co2+
Cu2+
Fe2+
HNO
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nitrosyl hydride replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase resulting in the incorporation of both N and O atoms into the product. Turnover is demonstrated by consumption of quercetin and other related substrates under anaerobic conditions in the presence of HNO-releasing compounds and the enzyme. As with dioxygenase activity, a nonenzymatic base-catalyzed reaction of quercetin with HNO isobserved above pH 7, but no enhancement of this basal reactivity is found upon addition of divalent metal salts. Unique and regioselective N-containing products are characterized by MS analysis for both the enzymatic and nonenzymatic reactions
Mn2+
Ni2+
additional information
Co2+
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Co2+ salt addition increases the activity of quercetin 2,3-dioxygenase 24fold. The Escherichia coli cultures were grown at 37°C and 200 rpm for 6 h, induced with isopropyl beta-D-thiogalactopyanoside to a final concentraton of 50 mg/l in the presence of 10 microM CoCl2, and allow to grow additional 4 h at 25°C. The protein contains 0.65-0.8 atom of cobalt and 0.1 atom of iron per subunit.
Cu2+
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Cu2+ salt addition increases the activity of quercetin 2,3-dioxygenase 1.4fold. The Escherichia coli cultures were grown at 37°C and 200 rpm for 6 h, induced with isopropyl beta-D-thiogalactopyanoside to a final concentraton of 50 mg/l in the presence of 10 microM CuCl2, and allow to grow additional 4 h at 25°C.
Cu2+
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activates, Cu-QDO, during the reaction mechanism of Cu-QDO dioxygen binds to the metal ion of the Cu-QDO-quercetin complex, yielding a Cu2+-superoxo quercetin radical intermediate, which then forms a Cu2+-alkylperoxo complex, the alkylperoxo complex evolves into endoperoxide intermediate that decomposes to the product
Mn2+
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Mn2+ salt addition increases the activity of quercetin 2,3-dioxygenase 35fold. The Escherichia coli cultures were grown at 37°C and 200 rpm for 6 h, induced with isopropyl beta-D-thiogalactopyanoside to a final concentraton of 50 mg/l in the presence of 10 microM MnSO4, and allow to grow additional 4 h at 25°C. The protein containes 1.6-1.9 atoms of Mn/subunit.
Mn2+
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activates, Mn-QDO, Mn2+ i the preferred metal ion. Mn-QDO in absence of O2 shows ability to react with nitroxyl (HNO)-singly reduced form of NO. HNO is incorporated into quercetin in the same manner as dioxygen, yet the reaction is strictly regioselective, as the only product is 2-((3,4-dihydroxyphenyl)(imino) methoxy)-4,6-dihydroxybenzoate
Ni2+
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Ni2+ salt addition increases the activity of quercetin 2,3-dioxygenase 2.6fold. The Escherichia coli cultures were grown at 37°C and 200 rpm for 6 h, induced with isopropyl beta-D-thiogalactopyanoside to a final concentraton of 50 mg/l in the presence of 10 microM NiCl2, and allow to grow additional 4 h at 25°C.
additional information
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Cd2+ does not increase the activity of quercetin 2,3-dioxygenase. The Escherichia coli cultures were grown at 37°C and 200 rpm for 6 h, induced with isopropyl beta-D-thiogalactopyanoside to a final concentraton of 50 mg/l in the presence of 10 microM CdCl2, and allow to grow additional 4 h at 25°C.
additional information
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Fe2+ does not increase the activity of quercetin 2,3-dioxygenase. The Escherichia coli cultures were grown at 37°C and 200 rpm for 6 h, induced with isopropyl beta-D-thiogalactopyanoside to a final concentraton of 50 mg/l in the presence of 10 microM FeCl2, and allow to grow additional 4 h at 25°C.
additional information
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Zn2+ does not increase the activity of quercetin 2,3-dioxygenase. The Escherichia coli cultures were grown at 37°C and 200 rpm for 6 h, induced with isopropyl beta-D-thiogalactopyanoside to a final concentraton of 50 mg/l in the presence of 10 microM ZnSO4, and allow to grow additional 4 h at 25°C.
additional information
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the bacterial enzyme is capable of using different divalent metal ions for catalysis, with preference Mn2+, Co2+, Fe2+, Ni2+, Cu2+in descending order, suggesting that the redox properties of the metal are relatively unimportant for the catalytic reaction. The major role of the active site metal ion could be to correctly position the substrate and to stabilize transition states and intermediates rather than to mediate electron transfer. The recombinant enzyme is able to exchange its active-site metal ion while retaining catalytic activity
additional information
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the enzyme from Bacillus subtilis is active with several divalent metal cofactors such as Fe, Mn, and Co, although Mn(II) is the preferred cofactor for this enzyme
additional information
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QDO is a mononuclear metalloenzyme hosting various transition metal ions (Cu2+, Mn2+, Fe2+) in its active site depending on the origin of the protein, different metal complex structures, overview
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
DTT
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Mn QueD: 96% activity remains after 15 min incubation with 1 mM reagent
H2O2
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Co QueD: 93% activity remains after 15 min incubation with 1 mM reagent, Mn QueD: 82% activity remains after 15 min incubation with 1 mM reagent
NaCN
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Co QueD: 91% activity remains after 15 min incubation with 1 mM reagent, Mn QueD: 89% activity remains after 15 min incubation with 1 mM reagent
O-ethylxanthic acid
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Co QueD: 69% activity remains after 15 min incubation with 1 mM reagent, Mn QueD: 100% activity remains after 15 min incubation with 1 mM reagent
Sodium ascorbate
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Mn QueD: 80% activity remains after 15 min incubation with 1 mM reagent
diethyldithiocarbamate
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Co QueD: 90% activity remains after 15 min incubation with 1 mM reagent, Mn QueD: 84% activity remains after 15 min incubation with 1 mM reagent
EDTA
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Mn QueD: 90% activity remains after 15 min incubation with 1 mM reagent
kojic acid
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Co QueD: 92% activity remains after 15 min incubation with 1 mM reagent, Mn QueD: 100% activity remains after 15 min incubation with 1 mM reagent
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
DTT
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Co QueD: 110% activity remains after 15 min incubation with 1 mM reagent
EDTA
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Co QueD: 110% activity remains after 15 min incubation with 1 mM reagent
NaN3
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Co QueD: 110% activity remains after 15 min incubation with 1 mM reagent, Mn QueD = 100% activity remains after 15 min incubation with 1 mM reagent
Sodium ascorbate
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Co QueD: 110% activity remains after 15 min incubation with 1 mM reagent
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
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kinetics, modeling, overview
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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
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
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pH profiles are determined spectrophotometrically on a Hp 8453 photodiode array spectrophotometer
additional information
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nonenzymatic, basal reactivity of sodium trioxodinitrate/Na2N2O3 (Angeli's salt) as HNO donor with quercetin initiates above pH 7.0
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
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pKa determined from the Vmax profile is 5.6. The pKa determined from the Vmax/KM pH profile is 5.8.
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
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the ring-cleaving dioxygenase belongs to the cupin superfamily, characterized by a six-stranded beta-barrel fold and conserved amino acid motifs that provide the 3His or 2- or 3His-1Glu ligand environment of a divalent metal ion. The cupin domain comprises two conserved amino acid motifs with the consensus sequences G(X)5HXH(X)3-4E(X)6G (motif 1) and G(X)5-7PXG(X)2H(X)3N
physiological function
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quercetin 2,3-dioxygenase (QDO) is an enzyme which accepts various transition metal ions as cofactors, and cleaves the heterocyclic ring of quercetin with consumption of dioxygen and release of carbon monoxide. QDO from Bacillus subtilis that binds Mn(II) displays an unprecedented nitroxygenase activity, whereby nitroxyl (HNO) is incorporated into quercetin cleavage products instead of dioxygen. The reaction proceeds with high regiospecificity, i.e. nitrogen and oxygen atoms of HNO are incorporated into specific fragments of the cleavage product. The reaction is an inherent property of the reactants, whereas the unique reactivity of Mn-QDO, as opposed to Co- or Fe-QDO that do not catalyze nitroxygenation. A nonenzymatic base-catalyzed reaction, which occurs in pH above 7.5, yields the same reaction products
additional information
additional information
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enzyme-bound quercetin shields the FeII cofactor from interactions with the O2 mimic nitric oxide, tentatively suggesting that the reaction catalyzed by Bacillus (Fe-)quercetinase may proceed without direct interaction of dioxygen and metal ion, overview
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
storage in buffer, loss of 25% of activity during 48 h, high concentrations of ammonium chloride stabilize, ascorbic acid and cysteine do not
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
DEAE-Sephacel column equilibrated with 50 mM Tris.HCl, pH 7.5, and eluted with a NaCl gradient (0-600 mM). Ultrogel ACA 34 column, eluted with 50 mM Tris.HCl, pH 7.5, and 100 mM Nacl. DEAE-Sepharose column equilibrated with 50 mM Tris.HCl, pH 7,5, eluted with a gradient of NaCl (100-500 mM).
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partial purification by ammonium sulfate precipitation and DEAE Toyopearl column chromatography
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Gopal, B.; Madan, L.L.; Betz, S.F.; Kossiakoff, A.A.
The crystal structure of a quercetin 2,3-dioxygenase from Bacillus subtilis suggests modulation of enzyme activity by a change in the metal ion at the active site(s)
Biochemistry
44
193-201
2005
Bacillus subtilis (P42106), Bacillus subtilis
Barney, B.M.; Schaab, M.R.; LoBrutto, R.; Francisco, W.A.
Evidence for a new metal in a known active site: purification and characterization of an iron-containing quercetin 2,3-dioxygenase from Bacillus subtilis
Protein Expr. Purif.
35
131-141
2004
Bacillus subtilis
Schaab, M.R.; Barney, B.M.; Francisco, W.A.
Kinetic and spectroscopic studies on the quercetin 2,3-dioxygenase from Bacillus subtilis
Biochemistry
45
1009-1016
2006
Bacillus subtilis
Hirooka, K.; Fujita, Y.
Excess production of Bacillus subtilis quercetin 2,3-dioxygenase affects cell viability in the presence of quercetin
Biosci. Biotechnol. Biochem.
74
1030-1038
2010
Bacillus subtilis, Bacillus subtilis 168
Fetzner, S.
Ring-cleaving dioxygenases with a cupin fold
Appl. Environ. Microbiol.
78
2505-2514
2012
Aspergillus japonicus, Bacillus subtilis, Penicillium olsonii, Streptomyces sp., Streptomyces sp. FLA / DSM 41951
Kumar, M.; Zapata, A.; Ramirez, A.; Bowen, S.; Francisco, W.; Farmer, P.
Nitrosyl hydride (HNO) replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase
Proc. Natl. Acad. Sci. USA
108
18926-18931
2011
Bacillus subtilis
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