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Literature summary extracted from
- 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 .
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 1.14.19.8 | gene CYP161C2 or pntM, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 | Streptomyces arenae |
Crystallization (Commentary)
| EC Number | Crystallization (Comment) | Organism |
|---|---|---|
| 1.14.19.8 | 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
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 1.14.19.8 | F232A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Streptomyces arenae |
| 1.14.19.8 | F232G | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Streptomyces arenae |
| 1.14.19.8 | F232H | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Streptomyces arenae |
| 1.14.19.8 | F232L | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Streptomyces arenae |
| 1.14.19.8 | F232Y | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Streptomyces arenae |
| 1.14.19.8 | M77S | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Streptomyces arenae |
| 1.14.19.8 | M81A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Streptomyces arenae |
| 1.14.19.8 | M81C | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Streptomyces arenae |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 1.14.19.8 | additional information | - |
additional information | steady-state kinetics | Streptomyces arenae | |
| 1.14.19.8 | 0.012 | - |
pentalenolactone F | recombinant mutant F232L, pH 8.0, 30°C | Streptomyces arenae |  |
| 1.14.19.8 | 0.019 | - |
pentalenolactone F | recombinant mutant M77S, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.019 | - |
pentalenolactone F | recombinant mutant M81C, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.036 | - |
pentalenolactone F | recombinant wild-type enzyme, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.037 | - |
pentalenolactone F | recombinant mutant M81A, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.043 | - |
pentalenolactone F | recombinant mutant F232A, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.28 | - |
pentalenolactone F | recombinant mutant M81C-2-mercaptoethanol conjugate, pH 8.0, 30°C | Streptomyces arenae | |
Metals/Ions
| EC Number | Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|---|
| 1.14.19.8 | Iron | Fe3+/Fe4+ during catalysis, a cytochrome P450 enzyme | Streptomyces arenae | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 1.14.19.8 | Streptomyces arenae | E3VWI3 | - |
- |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 1.14.19.8 | 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
| EC Number | Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|---|
| 1.14.19.8 | pentalenolactone F + O2 + 2 reduced ferredoxin + 2 H+ = pentalenolactone + 2 oxidized ferredoxin + 2 H2O | molecular reaction mechanism, overview | Streptomyces arenae |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.14.19.8 | 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 | ? | - |
? | |
| 1.14.19.8 | 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
| EC Number | Subunits | Comment | Organism |
|---|---|---|---|
| 1.14.19.8 | More | structure analysis of free enzyme and ligand bound enzyme, detailed overview | Streptomyces arenae |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 1.14.19.8 | CYP161C2 | - |
Streptomyces arenae |
| 1.14.19.8 | pntM | - |
Streptomyces arenae |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 1.14.19.8 | 30 | - |
assay at | Streptomyces arenae |
Turnover Number [1/s]
| EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 1.14.19.8 | 0.000067 | - |
pentalenolactone F | recombinant mutant M81A, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.00032 | - |
pentalenolactone F | recombinant mutant M77S, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.00058 | - |
pentalenolactone F | recombinant mutant F232A, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.00142 | - |
pentalenolactone F | recombinant mutant M81C-2-mercaptoethanol conjugate, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.0015 | - |
pentalenolactone F | recombinant mutant F232L, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.00153 | - |
pentalenolactone F | recombinant mutant M81C, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.03 | - |
pentalenolactone F | recombinant wild-type enzyme, pH 8.0, 30°C | Streptomyces arenae | |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 1.14.19.8 | 8 | - |
assay at | Streptomyces arenae |
Cofactor
| EC Number | Cofactor | Comment | Organism | Structure |
|---|---|---|---|---|
| 1.14.19.8 | cytochrome P-450 | - |
Streptomyces arenae | |
| 1.14.19.8 | Ferredoxin | - |
Streptomyces arenae |
General Information
| EC Number | General Information | Comment | Organism |
|---|---|---|---|
| 1.14.19.8 | metabolism | the enzyme catalyzes the oxidative rearrangement in the final step of the sesquiterpenoid antibiotic pentalenolactone biosynthesis | Streptomyces arenae |
| 1.14.19.8 | 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]
| EC Number | kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 1.14.19.8 | 0.00027 | - |
pentalenolactone F | recombinant mutant M81A, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.0051 | - |
pentalenolactone F | recombinant mutant M81C-2-mercaptoethanol conjugate, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.013 | - |
pentalenolactone F | recombinant mutant F232A, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.017 | - |
pentalenolactone F | recombinant mutant M77S, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.081 | - |
pentalenolactone F | recombinant mutant M81C, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.125 | - |
pentalenolactone F | recombinant mutant F232L, pH 8.0, 30°C | Streptomyces arenae | |
| 1.14.19.8 | 0.83 | - |
pentalenolactone F | recombinant wild-type enzyme, pH 8.0, 30°C | Streptomyces arenae | |
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