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Literature summary extracted from

  • Takahashi, S.; Zhao, Y.; O'Maille, P.E.; Greenhagen, B.T.; Noel, J.P.; Coates, R.M.; Chappell, J.
    Kinetic and molecular analysis of 5-epiaristolochene 1,3-dihydroxylase, a cytochrome P450 enzyme catalyzing successive hydroxylations of sesquiterpenes (2005), J. Biol. Chem., 280, 3686-3696.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.14.14.149 dimethyl sulfoxide maximum conversion of 5-epiaristolochene to capsidiol activity is observed at final concentrations of 2-5% (v/v) dimethyl sulfoxide Nicotiana tabacum

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.14.149 expressed in WAT11 yeast strain Nicotiana tabacum

Protein Variants

EC Number Protein Variants Comment Organism
1.14.14.149 I468A the mutant produces significant amounts 1beta-hydroxy-5-epiaristolochene, but negligible amounts of capsidiol from 5-epiaristolochene Nicotiana tabacum
1.14.14.149 S368A the mutant retains its ability to fully convert 5-epiaristolochene to capsidiol, but the turnover rates for capsidiol formation is 3-13times lower compared with that of the wild type enzyme and is also able to convert 1beta-hydroxy-5-epiaristolochene to capsidiol with kcat values comparable with that of wild type enzyme, but with Km values for 1beta-hydroxy-5-epiaristolochene 4-6times greater compared with that of wild type Nicotiana tabacum
1.14.14.149 S368C the mutant exhibits wild type catalytic efficiency for 1beta-hydroxy-5-epiaristolochene biosynthesis, but is devoid of the successive hydroxylation activity for capsidiol biosynthesis Nicotiana tabacum
1.14.14.149 S368F devoid of any hydroxylase activity for 5-epiaristolochene or 1beta-hydroxy-5-epiaristolochene Nicotiana tabacum
1.14.14.149 S368I devoid of any hydroxylase activity for 5-epiaristolochene or 1beta-hydroxy-5-epiaristolochene Nicotiana tabacum
1.14.14.149 S368T the mutant retains its ability to fully convert 5-epiaristolochene to capsidiol, but the turnover rates for capsidiol formation is 3-13times lower compared with that of the wild type enzyme and is also able to convert 1beta-hydroxy-5-epiaristolochene to capsidiol with kcat values comparable with that of wild type enzyme, but with Km values for 1beta-hydroxy-5-epiaristolochene 4-6times greater compared with that of wild type Nicotiana tabacum
1.14.14.149 S368V the mutant exhibits wild type catalytic efficiency for 1beta-hydroxy-5-epiaristolochene biosynthesis, but is devoid of the successive hydroxylation activity for capsidiol biosynthesis, the mutant catalyzes the relative equal biosynthesis of 1beta-hydroxy-5-epiaristolochene, 2beta-hydroxy-5-epiaristolochene, and 3beta-hydroxy-5-epiaristolochene from 5-epiaristolochene with wild type efficiency and converts about 1.5% of these monohydroxylated products to their respective ketone forms Nicotiana tabacum

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.14.14.149 dimethyl sulfoxide conversion of 5-epiaristolochene to capsidiol activity is inhibited at concentrations above 10% (v/v) dimethyl sulfoxide Nicotiana tabacum

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.14.149 0.00174
-
 1beta-hydroxy-5-epiaristolochene wild type enzyme, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.0062
-
 1beta-hydroxy-5-epiaristolochene mutant enzyme S368A, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.00997
-
 1beta-hydroxy-5-epiaristolochene mutant enzyme S368T, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.01518
-
 5-epiaristolochene mutant enzyme S368T, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.01672
-
 5-epiaristolochene mutant enzyme S386V, the reaction product is 1beta-hydroxy-5-epiaristolochene, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.01918
-
 5-epiaristolochene wild type enzyme, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.02124
-
 1beta-hydroxy-5-epiaristolochene mutant enzyme I486A, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.02267
-
 5-epiaristolochene mutant enzyme S368A, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.03863
-
 5-epiaristolochene mutant enzyme S386C, the reaction product is 1beta-hydroxy-5-epiaristolochene, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.0688
-
 5-epiaristolochene mutant enzyme I486A, the reaction product is 1beta-hydroxy-5-epiaristolochene, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum

Organism

EC Number Organism UniProt Comment Textmining
1.14.14.149 Nicotiana tabacum Q94FM7
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.14.149 1beta-hydroxy-5-epiaristolochene + [reduced NADPH-hemoprotein reductase] + O2 the catalytic efficiency for 1beta-hydroxy-5-epiaristolochene is about 10times greater than that for 3alpha-hydroxy-5-epiaristolochene Nicotiana tabacum capsidiol + [oxidized NADPH-hemoprotein reductase] + H2O
-
 ?
1.14.14.149 3alpha-hydroxy-5-epiaristolochene + [reduced NADPH-hemoprotein reductase] + O2 the catalytic efficiency for 3alpha-hydroxy-5-epiaristolochene is about 10times lower than that for 1beta-hydroxy-5-epiaristolochene Nicotiana tabacum capsidiol + [oxidized NADPH-hemoprotein reductase] + H2O
-
 ?
1.14.14.149 5-epiaristolochene + 2 [reduced NADPH-hemoprotein reductase] + 2 O2
-
 Nicotiana tabacum capsidiol + 2 [oxidized NADPH-hemoprotein reductase] + 2 H2O
-
 ?
1.14.14.149 5-epiaristolochene + [reduced NADPH-hemoprotein reductase] + O2 the release of an 1beta-hydroxy-5-epiaristolochene intermediate at high 5-epiaristolochene concentrations and a 10fold catalytic preference for 1beta-hydroxy-5-epiaristolochene versus 3alpha-hydroxy-5-epiaristolochene is indicative of a preferred reaction order of hydroxylation at C-1, followed by that at C-3 Nicotiana tabacum 1beta-hydroxy-5-epiaristolochene + [oxidized NADPH-hemoprotein reductase] + H2O
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.14.14.149 EAH
-
 Nicotiana tabacum

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.14.149 0.037
-
 5-epiaristolochene mutant enzyme S368T, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.132
-
 1beta-hydroxy-5-epiaristolochene mutant enzyme I486A, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.154
-
 5-epiaristolochene mutant enzyme S368A, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.278
-
 5-epiaristolochene mutant enzyme S386C, the reaction product is 1beta-hydroxy-5-epiaristolochene, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.293
-
 5-epiaristolochene mutant enzyme I486A, the reaction product is 1beta-hydroxy-5-epiaristolochene, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.482
-
 1beta-hydroxy-5-epiaristolochene mutant enzyme S368T, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.493
-
 5-epiaristolochene wild type enzyme, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.553
-
 5-epiaristolochene mutant enzyme S386V, the reaction product is 1beta-hydroxy-5-epiaristolochene, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.581
-
 1beta-hydroxy-5-epiaristolochene mutant enzyme S368A, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 0.582
-
 1beta-hydroxy-5-epiaristolochene wild type enzyme, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.14.149 heme
-
 Nicotiana tabacum

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.14.149 2.4
-
 5-epiaristolochene mutant enzyme S368T, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 4.3
-
 5-epiaristolochene mutant enzyme I486A, the reaction product is 1beta-hydroxy-5-epiaristolochene, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 6.2
-
 1beta-hydroxy-5-epiaristolochene mutant enzyme I486A, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 6.8
-
 5-epiaristolochene mutant enzyme S368A, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 7.2
-
 5-epiaristolochene mutant enzyme S386C, the reaction product is 1beta-hydroxy-5-epiaristolochene, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 25.7
-
 5-epiaristolochene wild type enzyme, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 33.1
-
 5-epiaristolochene mutant enzyme S386V, the reaction product is 1beta-hydroxy-5-epiaristolochene, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 48.3
-
 1beta-hydroxy-5-epiaristolochene mutant enzyme S368T, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 93.7
-
 1beta-hydroxy-5-epiaristolochene mutant enzyme S368A, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum
1.14.14.149 334.4
-
 1beta-hydroxy-5-epiaristolochene wild type enzyme, the reaction product is capsidiol, in 100 mM Tris-HCl (pH 7.5), at 30°C Nicotiana tabacum