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Literature summary extracted from

  • Green, S.; Squire, C.J.; Nieuwenhuizen, N.J.; Baker, E.N.; Laing, W.
    Defining the potassium binding region in an apple terpene synthase (2009), J. Biol. Chem., 284, 8661-8669.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.3.15 mutated enzymes are generated using the QuickChange II site-directed mutagenesis kit. The PCR-based mutagenesis protocol is performed using pET200-D-TOPO harbouring the respective AcTPS2 cDNAs as template. The single-site mutant enzymes overexpressed in Escherichia coli. Actinidia chinensis
4.2.3.46 mutated enzymes are generated using the QuickChange II site-directed mutagenesis kit. The PCR-based mutagenesis protocol is performed using pET-30a harbouring the MdAFS1 cDNAs as template. The single-site mutant enzymes overexpressed in Escherichia coli. Malus domestica

Protein Variants

EC Number Protein Variants Comment Organism
4.2.3.15 K514A AcTPS2 mutant, 90% reduction in monoterpene synthase activity compared to wild-type enzyme Actinidia chinensis
4.2.3.15 K514S AcTPS2 mutant, presence of Ser is not able to rescue any of the activity lost after the removal of the Lys residue Actinidia chinensis
4.2.3.46 D484A 85% loss of sesquiterpene synthase activity compared with wild-type enzyme when K+ is present Malus domestica
4.2.3.46 D484A 85% loss of sesquiterpene synthase activity compared with wild-type enzyme. Little change in K+-independent activity Malus domestica
4.2.3.46 S485A exhibits marginally increased sesquiterpene synthase activities, and an approximate 2fold increase in monoterpene synthase activity compared with the wild-type enzyme Malus domestica
4.2.3.46 S485A mutant exhibits marginally increased sesquiterpene synthase activities, and an approximate 2fold increase in monoterpene synthase activity compared with the wild-type enzyme Malus domestica
4.2.3.46 S487A 95% decrease in sesquiterpene synthase activity compared with wild-type enzyme when K+ is present Malus domestica
4.2.3.46 S487A 95% decrease in sesquiterpene synthase activity compared with wild-type enzyme. Little change in K+-independent activity Malus domestica
4.2.3.46 S487K The S487K mutant has 35-45% of the wild-type activity with farnesyl diphosphate, with no significant alterations in the alpha-farnesene isomer ratios produced and a small decrease in catalytic efficiency Malus domestica
4.2.3.46 S487K mutant has 35–45% of the wild-type activity with farnesyl diphosphate, with no significant alterations in the alpha-farnesene isomer ratios produced and a small decrease in catalytic efficiency Malus domestica
4.2.3.46 S488A mutant shows decreases in both sesqui- and monoterpene synthase activities compared with the WT enzyme, with mono-TPS activity being reduced more than sesquiterpene synthase activity. Sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced. Malus domestica
4.2.3.46 S488A mutant shows decreases in both sesqui- and mono-terpene synthases activities, compared with the wild-type enzyme, with mono-terpene synthases activity being reduced more than sesqui-terpene synthases activity Malus domestica

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.2.3.15 Mg2+ 10 mM are included in farnesyl diphosphate activity assay Actinidia chinensis
4.2.3.15 additional information wild-type AcTPS2 is significantly more active when KCl is absent from the activity buffer Actinidia chinensis
4.2.3.46 K+ MdAFS1 retains up to 12% of its activity in the absence of K+, enzyme contains K+ binding region, MdAFS1 exhibits a type II K+ response, MdAFS1 is not absolutely dependent upon M+ its unequivocal classification as type I or type II K+ activated, or that of any other terpene synthases, will not be possibl, then type I enzymes can exhibit type II kinetics and vice versa. Malus domestica
4.2.3.46 K+ activity is dependent on K+, the potassium binding region is defined Malus domestica
4.2.3.46 Mg2+ 10 mM are included in farnesyl diphosphate activity assay Malus domestica

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.2.3.15 additional information Actinidia chinensis terpenes produced from the wild-type and mutant enzymes are identical, showing low levels of beta-myrcene from AcTPS2-K514A ?
-
 ?
4.2.3.46 (2E,6E)-farnesyl diphosphate Malus domestica commentary (3E,6E)-alpha-farnesene + diphosphate
-
 ?
4.2.3.46 additional information Malus domestica the monoterpene synthase ((E)-beta-ocimene and beta-myrcene) and sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
4.2.3.15 Actinidia chinensis
-
-
-
4.2.3.46 Malus domestica B2ZZ11
-
-
4.2.3.46 Malus domestica Q84LB2
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.3.15 recombinant AcTPS2 protein is extracted and purified Actinidia chinensis
4.2.3.46 recombinant MdAFS1protein is extracted and purified Malus domestica

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
4.2.3.46 additional information
-
 S487K mutant has 35-45% of the wild-type activity with farnesyl diphosphate with no significant alterations in the alpha-farnesene isomer ratios produced and a small decrease in catalytic efficiency (wild-type and S487K respective kcat/Km values of 17.5 and 13.3 mM/s) Malus domestica

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.3.15 additional information terpenes produced from the wild-type and mutant enzymes are identical, showing low levels of beta-myrcene from AcTPS2-K514A Actinidia chinensis ?
-
 ?
4.2.3.46 (2E,6E)-farnesyl diphosphate
-
 Malus domestica (3E,6E)-alpha-farnesene + diphosphate
-
 ?
4.2.3.46 (2E,6E)-farnesyl diphosphate commentary Malus domestica (3E,6E)-alpha-farnesene + diphosphate
-
 ?
4.2.3.46 geranyl diphosphate poor substrate Malus domestica (E)-beta-ocimene + beta-myrcene
-
 ?
4.2.3.46 additional information the monoterpene synthase ((E)-beta-ocimene and beta-myrcene) and sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced Malus domestica ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
4.2.3.15 AcTPS2
-
 Actinidia chinensis
4.2.3.15 myrcene synthase
-
 Actinidia chinensis
4.2.3.46 alpha-farnesene synthase
-
 Malus domestica
4.2.3.46 MdAFS1
-
 Malus domestica

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.3.15 30
-
 farnesyl diphosphate and geranyl diphosphate activity assay Actinidia chinensis
4.2.3.46 30
-
 farnesyl diphosphate and geranyl diphosphate activity assay Malus domestica

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.2.3.46 0.000005
-
 geranyl diphosphate mutant S487A Malus domestica
4.2.3.46 0.000005
-
 geranyl diphosphate pH 7.5, 30°C, mutant enzyme S487A Malus domestica
4.2.3.46 0.000034
-
 geranyl diphosphate mutant D484A Malus domestica
4.2.3.46 0.000034
-
 geranyl diphosphate pH 7.5, 30°C, mutant enzyme D484A Malus domestica
4.2.3.46 0.000052
-
 geranyl diphosphate mutant S488A Malus domestica
4.2.3.46 0.000052
-
 geranyl diphosphate pH 7.5, 30°C, mutant enzyme S488A Malus domestica
4.2.3.46 0.000281
-
 geranyl diphosphate wild-type Malus domestica
4.2.3.46 0.000281
-
 geranyl diphosphate pH 7.5, 30°C, wild-type enzyme Malus domestica
4.2.3.46 0.000588
-
 geranyl diphosphate mutant S485A Malus domestica
4.2.3.46 0.000588
-
 geranyl diphosphate pH 7.5, 30°C, mutant enzyme S485A Malus domestica
4.2.3.46 0.0026
-
 farnesyl diphosphate mutant S487A, 50 mM KCl, 10 mM MgCl2 Malus domestica
4.2.3.46 0.0026
-
 (2E,6E)-farnesyl diphosphate pH 7.5, 30°C, mutant enzyme S487A Malus domestica
4.2.3.46 0.0062
-
 farnesyl diphosphate without K+ Malus domestica
4.2.3.46 0.0086
-
 farnesyl diphosphate mutant D484A, 50 mM KCl, 10 mM MgCl2 Malus domestica
4.2.3.46 0.0086
-
 (2E,6E)-farnesyl diphosphate pH 7.5, 30°C, mutant enzyme D484A Malus domestica
4.2.3.46 0.022
-
 farnesyl diphosphate without K+, sesquiterpene synthase activity in mutant S487K is independent of K+ Malus domestica
4.2.3.46 0.039
-
 farnesyl diphosphate mutant S488A, 50 mM KCl, 10 mM MgCl2 Malus domestica
4.2.3.46 0.039
-
 (2E,6E)-farnesyl diphosphate pH 7.5, 30°C, mutant enzyme S488A Malus domestica
4.2.3.46 0.0533
-
 farnesyl diphosphate wild-type MdAFS, 50 mM KCl, 10 mM MgCl2 Malus domestica
4.2.3.46 0.0553
-
 (2E,6E)-farnesyl diphosphate pH 7.5, 30°C, wild-type enzyme Malus domestica
4.2.3.46 0.0613
-
 farnesyl diphosphate mutant S485A, 50 mM KCl, 10 mM MgCl2 Malus domestica
4.2.3.46 0.0613
-
 (2E,6E)-farnesyl diphosphate pH 7.5, 30°C, mutant enzyme S485A Malus domestica

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.3.15 7.5
-
 farnesyl diphosphate activity assay Actinidia chinensis
4.2.3.46 7.5
-
 farnesyl diphosphate activity assay Malus domestica