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Information on EC 1.14.13.172 - salicylate 5-hydroxylase
for references in articles please use BRENDA:EC1.14.13.172
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EC Tree 1 Oxidoreductases
1.14 Acting on paired donors, with incorporation or reduction of molecular oxygen
1.14.13 With NADH or NADPH as one donor, and incorporation of one atom of oxygen into the other donor
1.14.13.172 salicylate 5-hydroxylase
IUBMB Comments
This enzyme, which was characterized from the bacterium Ralstonia sp. U2, comprises a multicomponent system, containing a reductase that is an iron-sulfur flavoprotein (FAD; EC 1.18.1.7, ferredoxin—NAD(P)+ reductase), an iron-sulfur oxygenase, and ferredoxin.
The enzyme appears in viruses and cellular organisms
- 1.14.13.172
- gentisate
- naphthalene
- ralstonia
- ferredoxin
- putida
- 2-hydroxybenzoate
- protocatechuate
-
naphthalene-degrading
-
polycyclic
- maleylpyruvate
-
rieske-type
- 1,2-dihydroxynaphthalene
-
nonheme
- 2-chlorobenzoate
- transposase
- fumarylpyruvate
-
cis-dihydrodiol
-
marr-type
- 1-hydroxylase
-
rieske
-
ring-cleavage
-
incp-7
- naphthalenivorans
- salicylaldehyde
-
chlorocatechol
-
polaromonas
showSynonyms
salicylate 5-hydroxylase, naggh, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
nagG
NagGH
nagH
S5H
SAL5H
nagG
-
-
-
-
nagH
-
-
-
-
S5H
a three-component system consisting of NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin)
S5H
a three-component system consisting of NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin)
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
salicylate + NADH + H+ + O2 = 2,5-dihydroxybenzoate + NAD+ + H2O
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
MetaCyc
salicylate degradation II, salicylate glucosides biosynthesis II
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SYSTEMATIC NAME
IUBMB Comments
salicylate,NADH:oxygen oxidoreductase (5-hydroxylating)
This enzyme, which was characterized from the bacterium Ralstonia sp. U2, comprises a multicomponent system, containing a reductase that is an iron-sulfur flavoprotein (FAD; EC 1.18.1.7, ferredoxin---NAD(P)+ reductase), an iron-sulfur oxygenase, and ferredoxin.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
anthranilate + NADH + H+ + O2
5-hydroxyanthranilate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
2,4-dihydroxybenzoate + NADH + H+ + O2
?
Substrates: -
Products: -
Products: -
?
2,4-dihydroxybenzoate + NADH + H+ + O2
?
Substrates: -
Products: -
Products: -
?
2,6-dihydroxybenzoate + NADH + H+ + O2
?
Substrates: -
Products: -
Products: -
?
2,6-dihydroxybenzoate + NADH + H+ + O2
?
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
-
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
-
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
-
Substrates: -
Products: -
Products: -
?
salicylate + NADPH + H+ + O2
2,5-dihydroxybenzoate + NADP+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADPH + H+ + O2
2,5-dihydroxybenzoate + NADP+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADPH + H+ + O2
2,5-dihydroxybenzoate + NADP+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADPH + H+ + O2
2,5-dihydroxybenzoate + NADP+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADPH + H+ + O2
2,5-dihydroxybenzoate + NADP+ + H2O
-
Substrates: -
Products: -
Products: -
?
salicylate + NADPH + H+ + O2
2,5-dihydroxybenzoate + NADP+ + H2O
-
Substrates: -
Products: -
Products: -
?
additional information
?
-
Substrates: 2-aminobenzoate might be a substrate for the enzyme
Products: -
Products: -
?
additional information
?
-
Substrates: no activity is detected against 2-hydroxycinnamate, 3-hydroxycinnamate, 2-hydroxyphenylacetate, 3-hydroxyphenylacetate, 2-hydroxybenzophenone, 1-hydroxy-2-naphthoate, 4-methoxysalicylate, and 2-hydroxyacetophenone
Products: -
Products: -
?
additional information
?
-
-
Substrates: no activity is detected against 2-hydroxycinnamate, 3-hydroxycinnamate, 2-hydroxyphenylacetate, 3-hydroxyphenylacetate, 2-hydroxybenzophenone, 1-hydroxy-2-naphthoate, 4-methoxysalicylate, and 2-hydroxyacetophenone
Products: -
Products: -
?
additional information
?
-
Substrates: no activity is detected against 2-hydroxycinnamate, 3-hydroxycinnamate, 2-hydroxyphenylacetate, 3-hydroxyphenylacetate, 2-hydroxybenzophenone, 1-hydroxy-2-naphthoate, 4-methoxysalicylate, and 2-hydroxyacetophenone
Products: -
Products: -
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADPH + H+ + O2
2,5-dihydroxybenzoate + NADP+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADPH + H+ + O2
2,5-dihydroxybenzoate + NADP+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADPH + H+ + O2
2,5-dihydroxybenzoate + NADP+ + H2O
Substrates: -
Products: -
Products: -
?
salicylate + NADPH + H+ + O2
2,5-dihydroxybenzoate + NADP+ + H2O
Substrates: -
Products: -
Products: -
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
SalABCD requires NAD(P)H as the cofactor instead of BH4
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Iron
Iron
the enzyme contains one Rieske-type [2Fe-2S] cluster and 7.5 g iron per mol enzyme
Iron
the reactive species that initiates catalysis appears to be an Fe(III)-superoxo species (or its electronic equivalent) which is made more reactive by the subsequent rapid transfer of an electron from the Rieske cluster. This transfer promotes O-O bond cleavage and drives the product forming reaction to completion
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.9398
2,6-dihydroxybenzoate
in 50 mM MES buffer (pH 6.4), at 22Ā°C
0.0029
salicylate
23Ā°C, pH not specified in the publication
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.64
salicylate
4Ā°C, pH not specified in the publication
1.86
salicylate
23Ā°C, pH not specified in the publication
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
107.1
after 124fold purification, in 50 mM MES buffer (pH 6.4), at 22Ā°C
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
large oxygenase component and small oxagenase component
UniProt
brenda
large oxygenase component and small oxagenase component
UniProt
brenda
large oxygenase component and small oxagenase component
UniProt
brenda
large oxygenase component and small oxagenase component
UniProt
brenda
O52378 i.e. reductase component NagAa, O52381 i.e. ferredoxin component NagAb cf. EC 1.18.1.7, O52379 i.e. large oxygenase component NagH, O52380 i.e. small oxygenase component NagH
UniProt
brenda
O52378 i.e. reductase component NagAa, O52381 i.e. ferredoxin component NagAb, O52379 i.e. large oxygenase component NagH, O52380 i.e. small oxygenase component NagH
UniProt
brenda
O52379 i.e. large oxygenase component NagG, O52380 i.e. small oxygenase component NagH
UniProt
brenda
Highest Expressing Human Cell Lines
Cell Line LinksPlease wait a moment until the data is sorted. This message will disappear when the data is sorted.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
binding of salicylate and O2 is fast. Transfer of the Rieske electron required to form the gentisate product occurs through bonds over about 12 A and must also be very fast. The observed rate constant for this reaction is much slower than expected and sensitive to substrate type. A transient intermediate (lambdamax of 700 nm) with an EPR resonance at g 4.3 is observed after product is formed in the active site. Gentisate binds to the mononuclear iron via its C5-OH in the intermediate. Rereduction of the metal centers accelerates product release about 300fold
physiological function
enzyme S5H hydroxylates salicylate into gentisate and is involved in the degradation of aromatic compounds. SalD is an essential component of the S5H complex
Show AA Sequence and Transmembrane Helices (188 entries)
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Please use the AA Sequence and Transmembrane Helices Search for a specific query.
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
18800
48799
x * 48799 + x * 18800, calculated from amino acid sequence
51000
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence
18800
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence
18800
x * 48799 + x * 18800, calculated from amino acid sequence
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
heterohexamer
homotetramer
heterohexamer
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence
heterohexamer
3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE
heterohexamer
-
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence
-
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
structure of the oxygenase component NagGH. The large subunit NagG and small subunit NagH share the same fold as that for their counterparts of Rieske dioxygenases and assemble the same alpha3beta3 hexamer. A potential substrate-binding pocket is observed in the vicinity of the nonheme iron site. Its positively charged residue Arg323 is surrounded by hydrophobic residues. The shift of nonheme iron atom caused by residue Leu228 disrupts the usual substrate pocket observed in other Rieske oxygenases. Residue Asn218 is involved in substrate binding and oxidation, but residues Gln316 and Ser367 play a more important role in substrate oxidation than Asn218
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
precursor-directed biosynthesis of 5-hydroxytryptophan using metabolically engineered Escherichia coli and involving the salicylate 5-hydroxylase from Ralstonia eutropha strain H16
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
the three-component Fe-S protein complex (NagAaGHAb) of the enzyme is purified by HisTrap column chromatography and Sephadex G-25 gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
enzyme S5H is encoded in the salABCD gene cluster, which locates on the chromosome rather than on a mobile plasmid, functional recombinant expression in Escherichia coli
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
biosynthetic pathway for maleate production in engineered Escherichia coli. Expression of salicylate 5-hydroxylase, introduction of salicylate biosynthetic pathway and gentisate ring cleavage pathway allow the synthesis of maleate from glycerol. Further optimizations boost maleate titer to 2.4 g/l in shake flask experiments. Subsequent scale-up biosynthesis of maleate in a 3-l bioreactor under fed-batch culture conditions enables the production of 14.5 g/l of maleate
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Suemori, A.; Nakajima, K.; Kurane, R.; Nakamura, Y.
Physicochemical and immunochemical characterization of salicylate 5-hydroxylase, m-hydroxybenzoate 6-hydroxylase and p-hydroxybenzoate 3-hydroxylase from Rhodococcus erythropolis
Biotechnol. Lett.
17
1063-1068
1995
Rhodococcus erythropolis, Rhodococcus erythropolis S1
-
brenda
Fang, T.; Zhou, N.Y.
Purification and characterization of salicylate 5-hydroxylase, a three-component monooxygenase from Ralstonia sp. strain U2
Appl. Microbiol. Biotechnol.
98
671-679
2014
Ralstonia sp. (O52379 and O52380), Ralstonia sp., Ralstonia sp. U2 (O52379 and O52380)
brenda
Fuenmayor, S.L.; Wild, M.; Boyes, A.L.; Williams, P.A.
A gene cluster encoding steps in conversion of naphthalene to gentisate in Pseudomonas sp. strain U2
J. Bacteriol.
180
2522-2530
1998
Pseudomonas sp. (O52379 and O52380), Pseudomonas sp., Pseudomonas sp. U2 (O52379 and O52380)
brenda
Zhou, N.Y.; Al-Dulayymi, J.; Baird, M.S.; Williams, P.A.
Salicylate 5-hydroxylase from Ralstonia sp. strain U2: a monooxygenase with close relationships to and shared electron transport proteins with naphthalene dioxygenase
J. Bacteriol.
184
1547-1555
2002
Ralstonia sp. (O52379 and O52380), Ralstonia sp., Ralstonia sp. U2 (O52379 and O52380)
brenda
Sun, X.; Lin, Y.; Yuan, Q.; Yan, Y.
Precursor-directed biosynthesis of 5-hydroxytryptophan using metabolically engineered E. coli
ACS Synth. Biol.
4
554-558
2015
Cupriavidus necator (Q0KB56)
brenda
Hou, Y.J.; Guo, Y.; Li, D.F.; Zhou, N.Y.
Structural and biochemical analysis reveals a distinct catalytic site of salicylate 5-monooxygenase NagGH from Rieske dioxygenases
Appl. Environ. Microbiol.
87
e01629
2021
Ralstonia sp. (O52378 and O52381 and O52379 and O52380), Ralstonia sp.
brenda
Rogers, M.S.; Lipscomb, J.D.
Salicylate 5-hydroxylase intermediates in aromatic hydroxylation by a Rieske monooxygenase
Biochemistry
58
5305-5319
2019
Ralstonia sp. (O52378 and O52381 and O52379 and O52380)
brenda
Sheng, H.; Jing, Y.; An, N.; Shen, X.; Sun, X.; Yan, Y.; Wang, J.; Yuan, Q.
Extending the shikimate pathway for microbial production of maleate from glycerol in engineered Escherichia coli
Biotechnol. Bioeng.
118
1840-1850
2021
Ralstonia sp. (O52379 and O52380)
brenda
EXTERNAL LINKS (specific for EC-Number 1.14.13.172)
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Release 2023.1 (January 2023)
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