Literature summary for 1.14.19.8 extracted from

Duan, L.; Jogl, G.; Cane, D.
The cytochrome P450-catalyzed oxidative rearrangement in the final step of pentalenolactone biosynthesis substrate structure determines mechanism (2016), J. Am. Chem. Soc., 138, 12678-12689 .

Search for more literature for 1.14.19.8

Cloned(Commentary)

Cloned (Comment)	Organism
gene CYP161C2 or pntM, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21	Streptomyces arenae

Crystallization (Commentary)

Crystallization (Comment)	Organism
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	Streptomyces arenae

Crystallization (Comment)

Organism

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

Streptomyces arenae

Protein Variants

Protein Variants	Comment	Organism
F232A	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Streptomyces arenae
F232G	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Streptomyces arenae
F232H	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Streptomyces arenae
F232L	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Streptomyces arenae
F232Y	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Streptomyces arenae
M77S	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Streptomyces arenae
M81A	site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme	Streptomyces arenae
M81C	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Streptomyces arenae

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	steady-state kinetics	Streptomyces arenae
0.012	-	pentalenolactone F	recombinant mutant F232L, pH 8.0, 30°C	Streptomyces arenae
0.019	-	pentalenolactone F	recombinant mutant M77S, pH 8.0, 30°C	Streptomyces arenae
0.019	-	pentalenolactone F	recombinant mutant M81C, pH 8.0, 30°C	Streptomyces arenae
0.036	-	pentalenolactone F	recombinant wild-type enzyme, pH 8.0, 30°C	Streptomyces arenae
0.037	-	pentalenolactone F	recombinant mutant M81A, pH 8.0, 30°C	Streptomyces arenae
0.043	-	pentalenolactone F	recombinant mutant F232A, pH 8.0, 30°C	Streptomyces arenae
0.28	-	pentalenolactone F	recombinant mutant M81C-2-mercaptoethanol conjugate, pH 8.0, 30°C	Streptomyces arenae

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Iron	Fe3+/Fe4+ during catalysis, a cytochrome P450 enzyme	Streptomyces arenae

Organism

Organism	UniProt	Comment	Textmining
Streptomyces arenae	E3VWI3	-	-

Purification (Commentary)

Purification (Comment)	Organism
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	Streptomyces arenae

Reaction

Reaction	Comment	Organism	Reaction ID
pentalenolactone F + O2 + 2 reduced ferredoxin + 2 H+ = pentalenolactone + 2 oxidized ferredoxin + 2 H2O	molecular reaction mechanism, overview	Streptomyces arenae	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	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	Streptomyces arenae	?	-	?
pentalenolactone F + O2 + 2 reduced ferredoxin + 2 H+	apparent ability of PntM to catalyze an exclusive carbocation-based oxidative rearrangement	Streptomyces arenae	pentalenolactone + 2 oxidized ferredoxin + 2 H2O	-	?

Subunits

Subunits	Comment	Organism
More	structure analysis of free enzyme and ligand bound enzyme, detailed overview	Streptomyces arenae

Synonyms

Synonyms	Comment	Organism
CYP161C2	-	Streptomyces arenae
pntM	-	Streptomyces arenae

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Streptomyces arenae

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.000067	-	pentalenolactone F	recombinant mutant M81A, pH 8.0, 30°C	Streptomyces arenae
0.00032	-	pentalenolactone F	recombinant mutant M77S, pH 8.0, 30°C	Streptomyces arenae
0.00058	-	pentalenolactone F	recombinant mutant F232A, pH 8.0, 30°C	Streptomyces arenae
0.00142	-	pentalenolactone F	recombinant mutant M81C-2-mercaptoethanol conjugate, pH 8.0, 30°C	Streptomyces arenae
0.0015	-	pentalenolactone F	recombinant mutant F232L, pH 8.0, 30°C	Streptomyces arenae
0.00153	-	pentalenolactone F	recombinant mutant M81C, pH 8.0, 30°C	Streptomyces arenae
0.03	-	pentalenolactone F	recombinant wild-type enzyme, pH 8.0, 30°C	Streptomyces arenae

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Streptomyces arenae

Cofactor

Cofactor	Comment	Organism	Structure
cytochrome P-450	-	Streptomyces arenae
Ferredoxin	-	Streptomyces arenae

General Information

General Information	Comment	Organism
metabolism	the enzyme catalyzes the oxidative rearrangement in the final step of the sesquiterpenoid antibiotic pentalenolactone biosynthesis	Streptomyces arenae
additional information	structure analysis of free enzyme and ligand bound enzyme, detailed overview. The topology of PntM undergoes minimal changes upon binding of ligands	Streptomyces arenae

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.00027	-	pentalenolactone F	recombinant mutant M81A, pH 8.0, 30°C	Streptomyces arenae
0.0051	-	pentalenolactone F	recombinant mutant M81C-2-mercaptoethanol conjugate, pH 8.0, 30°C	Streptomyces arenae
0.013	-	pentalenolactone F	recombinant mutant F232A, pH 8.0, 30°C	Streptomyces arenae
0.017	-	pentalenolactone F	recombinant mutant M77S, pH 8.0, 30°C	Streptomyces arenae
0.081	-	pentalenolactone F	recombinant mutant M81C, pH 8.0, 30°C	Streptomyces arenae
0.125	-	pentalenolactone F	recombinant mutant F232L, pH 8.0, 30°C	Streptomyces arenae
0.83	-	pentalenolactone F	recombinant wild-type enzyme, pH 8.0, 30°C	Streptomyces arenae