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Literature summary for 4.2.3.13 extracted from

  • Loizzi, M.; Gonzalez, V.; Miller, D.J.; Allemann, R.K.
    Nucleophilic water capture or proton loss single amino acid switch converts delta-cadinene synthase into germacradien-4-ol synthase (2018), ChemBioChem, 19, 100-105 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 4.2.3.13

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Gossypium arboreum
recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Gossypium arboreum

Protein Variants

Protein Variants Comment Organism
additional information if W279 in delta-cadinene synthase is replaced with various smaller amino acids, the ratio of alcohol versus hydrocarbon product is directly proportional to the van der Waals volume of the amino acid side chain Gossypium arboreum
N403P/L405H the double-mutant DCS converts farnesyl diphosphate into germacradien-4-ol (93%) and an additional unidentified cyclic sesquiterpene alcohol. The catalytic activity of DCS mutant N403P/L405H is severely compromised relative to that of the wild-type enzyme Gossypium arboreum
N403P/L405H the mutant enzyme converts (2E,6E)-farnesyl diphosphate into germacradien-4-ol (93%) and an additional unidentified cyclic sesquiterpene alcohol.However, the catalytic activity of the mutant is severely compromised relative to that of the wild type enzyme Gossypium arboreum
W279A site-directed mutagenesis, the mutant is a catalytically highly efficient germacradien-4-ol synthase that produces predominantly germacradien-4-ol in addition to 11% delta-cadinene Gossypium arboreum
W279A the mutant is a catalytically highly efficient germacradien-4-ol synthase that produces predominantly germacradien-4-ol (89%) in addition to 11% (+)-delta-cadinene Gossypium arboreum
W279D site-directed mutagenesis, the mutant shows altered substrate specificity and increased germacradien-4-ol synthase activity compared to the wild-type enzyme Gossypium arboreum
W279D the mutant produces 60% germacradien-4-ol and 40% (+)-delta-cadinene from (2E,6E)-farnesyl diphosphate Gossypium arboreum
W279E site-directed mutagenesis, the mutant shows altered substrate specificity and increased germacradien-4-ol synthase activity compared to the wild-type enzyme Gossypium arboreum
W279E the mutant produces 50% germacradien-4-ol and 50% (+)-delta-cadinene from (2E,6E)-farnesyl diphosphate Gossypium arboreum
W279L site-directed mutagenesis, the mutant shows altered substrate specificity and increased germacradien-4-ol synthase activity compared to the wild-type enzyme Gossypium arboreum
W279L the mutant produces 32% germacradien-4-ol and 68% (+)-delta-cadinene from (2E,6E)-farnesyl diphosphate Gossypium arboreum
W279M site-directed mutagenesis, the mutant shows altered substrate specificity and increased germacradien-4-ol synthase activity compared to the wild-type enzyme Gossypium arboreum
W279M the mutant produces 35% germacradien-4-ol and 65% (+)-delta-cadinene from (2E,6E)-farnesyl diphosphate Gossypium arboreum
W279Q site-directed mutagenesis, the mutant shows altered substrate specificity and increased germacradien-4-ol synthase activity compared to the wild-type enzyme Gossypium arboreum
W279Q the mutant produces 40% germacradien-4-ol and 60% (+)-delta-cadinene from (2E,6E)-farnesyl diphosphate Gossypium arboreum
W279Y site-directed mutagenesis, the mutant shows altered substrate specificity and increased germacradien-4-ol synthase activity compared to the wild-type enzyme Gossypium arboreum
W279Y the mutant produces 17% germacradien-4-ol and 83% (+)-delta-cadinene from (2E,6E)-farnesyl diphosphate Gossypium arboreum

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten steady-state kinetics, overview Gossypium arboreum
0.00058
-
 (2E,6E)-farnesyl diphosphate wild type enzyme, at pH 7.5 and 25°C Gossypium arboreum
0.00058
-
 (2E,6E)-farnesyl diphosphate recombinant His6-tagged wild-type enzyme, pH 7.5, 25°C Gossypium arboreum
0.00145
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279E, at pH 7.5 and 25°C Gossypium arboreum
0.00145
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279E, pH 7.5, 25°C Gossypium arboreum
0.0019
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279M, at pH 7.5 and 25°C Gossypium arboreum
0.0019
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279M, pH 7.5, 25°C Gossypium arboreum
0.0022
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279A, pH 7.5, 25°C Gossypium arboreum
0.00223
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279A, at pH 7.5 and 25°C Gossypium arboreum
0.0032
-
 (2E,6E)-farnesyl diphosphate wild type enzyme, at pH 7.5 and 25°C Gossypium arboreum
0.00428
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279Y, at pH 7.5 and 25°C Gossypium arboreum
0.0043
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279Y, pH 7.5, 25°C Gossypium arboreum
0.008
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279Q, at pH 7.5 and 25°C Gossypium arboreum
0.008
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279Q, pH 7.5, 25°C Gossypium arboreum
0.0092
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279D, pH 7.5, 25°C Gossypium arboreum
0.00924
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279D, at pH 7.5 and 25°C Gossypium arboreum
0.00945
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279L, at pH 7.5 and 25°C Gossypium arboreum
0.0095
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279L, pH 7.5, 25°C Gossypium arboreum

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Gossypium arboreum
Mg2+ required for catalysis Gossypium arboreum

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
(2E,6E)-farnesyl diphosphate Gossypium arboreum
-
 (+)-delta-cadinene + diphosphate
-
 ?
(2E,6E)-farnesyl diphosphate Gossypium arboreum
-
 (+)-delta-cadinene + germacradien-4-ol + diphosphate
-
 ?
additional information Gossypium arboreum delta-cadinene synthase is a sesquiterpene cyclase that utilises the universal achiral precursor farnesyl diphosphate (FDP) to generate predominantly the bicyclic sesquiterpene delta-cadinene and about 2% germacradien-4-ol ?
-
 ?

Organism

Organism UniProt Comment Textmining
Gossypium arboreum Q39761
-
-

Purification (Commentary)

Purification (Comment) Organism
Ni-NTA column chromatography Gossypium arboreum
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, diaylsis, and ultrafiltration Gossypium arboreum

Reaction

Reaction Comment Organism Reaction ID
(2E,6E)-farnesyl diphosphate = (+)-delta-cadinene + diphosphate catalytic mechanism, overview Gossypium arboreum
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(2E,6E)-farnesyl diphosphate
-
 Gossypium arboreum (+)-delta-cadinene + diphosphate
-
 ?
(2E,6E)-farnesyl diphosphate
-
 Gossypium arboreum (+)-delta-cadinene + germacradien-4-ol + diphosphate
-
 ?
additional information delta-cadinene synthase is a sesquiterpene cyclase that utilises the universal achiral precursor farnesyl diphosphate (FDP) to generate predominantly the bicyclic sesquiterpene delta-cadinene and about 2% germacradien-4-ol Gossypium arboreum ?
-
 ?

Synonyms

Synonyms Comment Organism
DCS
-
 Gossypium arboreum
Dcs1
-
 Gossypium arboreum
delta-cadinene synthase
-
 Gossypium arboreum

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Gossypium arboreum

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.00059
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279E, at pH 7.5 and 25°C Gossypium arboreum
0.00059
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279E, pH 7.5, 25°C Gossypium arboreum
0.00067
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279D, at pH 7.5 and 25°C Gossypium arboreum
0.00067
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279D, pH 7.5, 25°C Gossypium arboreum
0.00073
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279Y, at pH 7.5 and 25°C Gossypium arboreum
0.00073
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279Y, pH 7.5, 25°C Gossypium arboreum
0.001
-
 (2E,6E)-farnesyl diphosphate wild type enzyme, at pH 7.5 and 25°C Gossypium arboreum
0.00109
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279M, at pH 7.5 and 25°C Gossypium arboreum
0.0011
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279M, pH 7.5, 25°C Gossypium arboreum
0.00127
-
 (2E,6E)-farnesyl diphosphate His-tagged recombinant enzyme, at pH 7.5 and 25°C Gossypium arboreum
0.00127
-
 (2E,6E)-farnesyl diphosphate recombinant His6-tagged wild-type enzyme, pH 7.5, 25°C Gossypium arboreum
0.003
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279Q, at pH 7.5 and 25°C Gossypium arboreum
0.003
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279Q, pH 7.5, 25°C Gossypium arboreum
0.0031
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279A, pH 7.5, 25°C Gossypium arboreum
0.00312
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279A, at pH 7.5 and 25°C Gossypium arboreum
0.0038
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279L, at pH 7.5 and 25°C Gossypium arboreum
0.0038
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279L, pH 7.5, 25°C Gossypium arboreum

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Gossypium arboreum

General Information

General Information Comment Organism
malfunction if W279 in delta-cadinene synthase is replaced with various smaller amino acids, the ratio of alcohol versus hydrocarbon product is directly proportional to the van der Waals volume of the amino acid side chain. Mutant DCS-W279A is a catalytically highly efficient germacradien-4-ol synthase that produces predominantly germacradien-4-ol in addition to 11% delta-cadinene Gossypium arboreum
additional information catalytic mechanisms of (+)-delta-cadinene synthase (DCS) and germacradien-4-ol synthase (GdolS) catalysed conversions of (2E,6E)-farnesyl diphosphate to of (+)-delta-cadinene and germacradien-4-ol, analysis of the mechanism by which sesquiterpene synthases discriminate between deprotonation and reaction with a nucleophilic water molecule, molecular dynamics simulations, overview. Enzyme active site structure with bound Mg2+ ions Gossypium arboreum
physiological function enzyme DCS from Gossypium arboreum produces (+)-delta-cadinene in the first committed step of the biosynthetic pathway to the phytoalexin gossypol Gossypium arboreum

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.073
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279D, pH 7.5, 25°C Gossypium arboreum
0.17
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279Y, pH 7.5, 25°C Gossypium arboreum
0.38
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279Q, pH 7.5, 25°C Gossypium arboreum
0.4
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279L, pH 7.5, 25°C Gossypium arboreum
0.41
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279E, pH 7.5, 25°C Gossypium arboreum
0.58
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279M, pH 7.5, 25°C Gossypium arboreum
0.7
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279D, at pH 7.5 and 25°C Gossypium arboreum
1.41
-
 (2E,6E)-farnesyl diphosphate recombinant mutant W279A, pH 7.5, 25°C Gossypium arboreum
1.7
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279Y, at pH 7.5 and 25°C Gossypium arboreum
2.2
-
 (2E,6E)-farnesyl diphosphate recombinant His6-tagged wild-type enzyme, pH 7.5, 25°C Gossypium arboreum
2.5
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279Q, at pH 7.5 and 25°C Gossypium arboreum
4
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279L, at pH 7.5 and 25°C Gossypium arboreum
4.1
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279E, at pH 7.5 and 25°C Gossypium arboreum
5.7
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279M, at pH 7.5 and 25°C Gossypium arboreum
14
-
 (2E,6E)-farnesyl diphosphate mutant enzyme W279A, at pH 7.5 and 25°C Gossypium arboreum
21.7
-
 (2E,6E)-farnesyl diphosphate wild type enzyme, at pH 7.5 and 25°C Gossypium arboreum
31
-
 (2E,6E)-farnesyl diphosphate wild type enzyme, at pH 7.5 and 25°C Gossypium arboreum