Information on EC 1.14.19.8 - pentalenolactone synthase

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The expected taxonomic range for this enzyme is: Streptomyces

EC NUMBER
COMMENTARY hide
1.14.19.8
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RECOMMENDED NAME
GeneOntology No.
pentalenolactone synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
pentalenolactone F + O2 + 2 reduced ferredoxin + 2 H+ = pentalenolactone + 2 oxidized ferredoxin + 2 H2O
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
pentalenolactone biosynthesis
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Biosynthesis of antibiotics
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SYSTEMATIC NAME
IUBMB Comments
pentalenolactone-F:oxidized-ferredoxin oxidoreductase (pentalenolactone forming)
A heme-thiolate protein (P-450). Isolated from the bacteria Streptomyces exfoliatus and Streptomyces arenae.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
the enzyme catalyzes the oxidative rearrangement in the final step of the sesquiterpenoid antibiotic pentalenolactone biosynthesis
additional information
structure analysis of free enzyme and ligand bound enzyme, detailed overview. The topology of PntM undergoes minimal changes upon binding of ligands
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
pentalenolactone F + O2 + 2 reduced ferredoxin + 2 H+
pentalenolactone + 2 oxidized ferredoxin + 2 H2O
show the reaction diagram
pentalenolactone F + O2 + NADPH + H+
pentalenolactone + NADP+ + H2O
show the reaction diagram
additional information
?
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no activity with substrate analogue 6,7-dihydropentalenolactone, because the C1 carbocation is not anchimerically stabilized by the 6,7-double bond of pentalenolactone F. Enzyme-ligand interaction via three residues, F232, M77, and M81 that are unique to PntM and its orthologues and absent from essentially all other P450s
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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
pentalenolactone F + O2 + NADPH + H+
pentalenolactone + NADP+ + H2O
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome P-450
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NADPH
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Iron
Fe3+/Fe4+ during catalysis, a cytochrome P450 enzyme
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.012 - 0.43
pentalenolactone F
additional information
additional information
steady-state kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000067 - 10.5
pentalenolactone F
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00027 - 0.83
pentalenolactone F
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
UNIPROT
ORGANISM
Streptomyces arenae;
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
structure analysis of free enzyme and ligand bound enzyme, detailed overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified wild-type enzyme enzyme PntM substrate-free, and PntM with bound substrate pentalenolactone F, product pentalenolactone, and substrate analogue 6,7-dihydropentalenolactone, and recombinant PntM F232L, M77S, M81A, M81C, and M81C-BME mutants with bound pentalenolactone F, sitting drop vapor diffusion method, mixing of 0.001 ml of 16.8 mg/mL protein in 10 mM Tris-HCl, 15 mM NaCl, and 10% glycerol, with 0.001 ml of reservoir solution containing 1.2 M sodium citrate, 10% glycerol, and 100 mM bicine, pH 9.0, 15°C, followed by cross-microseeding technique, X-ray diffraction structure determination and analysis at 1.54 A and 2.06-2.12 A resolution, respectively, molecular replacement using the crystal structure of polyene macrolide epoxidase PimD, PDB ID 2X9P, as search model
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni2+-NTA resin column chromatography
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 by nickel affinity chromatography, tag cleavage by thrombin, another step of nickel affinity chromatography, and gel filtration of the eluate, followed by ultrafiltration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
gene CYP161C2 or pntM, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
transcription is activated by the PenR protein
transcription is activated by the PntR protein
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F232A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F232G
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F232H
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F232L
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F232Y
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
M77S
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
M81A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
M81C
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme