Information on EC 1.14.13.188 - 6-deoxyerythronolide B hydroxylase

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The expected taxonomic range for this enzyme is: Saccharopolyspora erythraea

EC NUMBER
COMMENTARY hide
1.14.13.188
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RECOMMENDED NAME
GeneOntology No.
6-deoxyerythronolide B hydroxylase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
6-deoxyerythronolide B + NADPH + H+ + O2 = erythronolide B + NADP+ + H2O
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of 12-, 14- and 16-membered macrolides
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Biosynthesis of antibiotics
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erythromycin D biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
6-deoxyerythronolide-B,NADPH:oxygen oxidoreductase
A heme-thiolate protein (P-450). Isolated from the bacterium Saccharopolyspora erythraea. The enzyme is involved in the biosynthesis of the antibiotic erythromycin.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(9R)-9-deoxo-9-hydroxy-6-deoxyerythronolide B + NADPH + H+ + O2
(9R)-9-deoxo-9-hydroxyerythronolide B + NADP+ + H2O
show the reaction diagram
(9S)-9-deoxo-9-hydroxy-6-deoxyerythronolide B + NADPH + H+ + O2
(9S)-9-deoxo-9-hydroxyerythronolide B + NADP+ + H2O
show the reaction diagram
(9S)-9-deoxo-9-hydroxy-8,8a-deoxyoleandolide + NADPH + H+ + O2
6-hydroxy-8,8a-deoxyoleandolide + NADP+ + H2O
show the reaction diagram
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substrate is identical to 6-deoxyerythronolide B except for the presence of a C13 methyl group. Hydroxylation at a rate approximately 4fold lower than the natural substrate 6-deoxyerythronolide B
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?
6-deoxyerythronolide B + NADPH + H+ + O2
erythronolide B + NADP+ + H2O
show the reaction diagram
8,8a-deoxyoleandolide + NADPH + H+ + O2
6-hydroxy-8,8a-deoxyoleandolide + NADP+ + H2O
show the reaction diagram
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substrate is identical to 6-deoxyerythronolide B except for the presence of a C13 methyl group. Hydroxylation at a rate approximately 4fold lower than the natural substrate 6-deoxyerythronolide B
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?
9-aminophenanthrene + NADPH + H+
? + NADP+ + H2O
show the reaction diagram
androstenedione + NADPH + H+
? + NADP+ + H2O
show the reaction diagram
dehydroepiandrostenedione + NADPH + H+
? + NADP+ + H2O
show the reaction diagram
testosterone + NADPH + H+
? + 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
6-deoxyerythronolide B + NADPH + H+ + O2
erythronolide B + NADP+ + H2O
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome P450
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heme
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a heme-thiolate protein (P-450)
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ketoconazole
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wild-type and mutant A245S show type-II binding. Mutant A245T shows type-I binding
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0115
(9R)-9-deoxo-9-hydroxy-6-deoxyerythronolide B
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pH 7.3, 35°C
0.0103 - 0.0312
9-aminophenanthrene
0.261 - 0.277
androstenedione
0.326
dehydroepiandrostenedione
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wild-type, pH 7.5, 37°C, S50 value, Hill coefficient 1.55
0.278
testosterone
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wild-type, pH 7.5, 37°C, S50 value, Hill coefficient 1.64
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.6
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isoelectric focusing
PDB
SCOP
CATH
ORGANISM
UNIPROT
Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338)
Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338)
Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338)
Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338)
Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338)
Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338)
Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338)
Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338)
Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 44000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
modeling of the substrate-binding pocket with ketoconazole bound shos that the secondary nitrogen of the azole ring is coordinated to the heme iron, and nearby regions are positioned in the active site similarly to 6-deoxyerythronolide B, the remainder of the molecule extends into the active site pocket, which is occupied by water in the 6-deoxyerythronolide B complex. The large water-binding pocket in the P450eryF active site appears to provide flexibility in substrate binding and allows for molecules that are larger than 6-deoxyerythronolide B to be accommodated in the active site
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molecular dynamics and hybrid quantum mechanics/molecular mechanics analysis. Two water networks exist around the active site, the one found in the crystal structure involving E360 and an alternative one involving E244. The first proton transfer that converts the peroxo to the hydroperoxo intermediate proceeds via the E244 water network with direct involvement of the 5-OH group of the substrate. For the second proton transfer, the computed barriers for the rate-limiting homolytic O-O cleavage are similar for the E360 and E244 pathways, and hence both glutamate residues may serve as proton source in this step
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NMR studies on lgad binding. Binding of 9-aminophenanthrene and testosterone occurs with apparent negative homotropic cooperativity for testosterone and positive homotropic cooperativity for 9-aminophenanthrene with Hill-equation-derived dissociation constants of 4 and 200 microM, respectively. Binding occurs on intermediate and fast chemical exhange time scales, respectively. The 15N-Phe NMR resonances most affected are the same in each titration, suggesting that the two ligands contact the same phenylalanines within the active site of P450eryF
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sitting drop vapor diffusion crystallization, crystal structures of the ferrous dioxygen complex of wild-type enzyme and its mutants, A245S and A245T
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to 2.2 A resolution. The substrate is positioned with the macrolide ring perpendicular to the heme plane and contacts seven hydrophobic residues and three solvent molecules. The substrate participates in a network of hydrogen bonds that may provide a proton shuttle pathway in the oxygen cleavage reaction
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
isolation of two forms, P-4501 and P-45011, by hydroxylapatite chromatography, having a P-450 content of 17.5 and 15.2 nmol/mg of protein, respectively
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A245S
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activity is decreased by 84%, the active site structure including water is essentially unchanged with the exception that the OH group of Ser245 points toward the I-helix cleft to make new H-bonds with water 63 and the carbonyl group of the Ala241; significantly increased activity with substrates testosterone and 7-benzyloxyquinoline. Contrary to wild-type, binding to inhibitor ketoconazole follows type II
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A245T
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increased activity with substrate 7-benzyloxyquinoline. Like wild-type, type-II binding to inhibitor ketoconazole; more than 99% loss of activity
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