Information on EC 4.2.3.9 - aristolochene synthase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY
4.2.3.9
-
RECOMMENDED NAME
GeneOntology No.
aristolochene synthase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
(2E,6E)-farnesyl diphosphate = aristolochene + diphosphate
show the reaction diagram
further cyclization and methyl transfer converts the intermediate into aristolochene
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
cyclization
-
-
internal cyclization
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
Metabolic pathways
-
Sesquiterpenoid and triterpenoid biosynthesis
-
SYSTEMATIC NAME
IUBMB Comments
(2E,6E)-farnesyl diphosphate diphosphate-lyase (cyclizing, aristolochene-forming)
The initial internal cyclization produces the monocyclic intermediate germacrene A; further cyclization and methyl transfer converts the intermediate into aristolochene. While in some species germacrene A remains as an enzyme-bound intermediate, it has been shown to be a minor product of the reaction in Penicillium roqueforti [5] (see also EC 4.2.3.23, germacrene-A synthase). The enzyme from Penicillium roqueforti requires Mg2+. Mn2+ can partially substitute, at low concentrations. Aristolochene is the likely parent compound for a number of sesquiterpenes produced by filamentous fungi.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5-epi-aristolochene
-
-
5-epi-aristolochene synthase
-
-
aristolochene synthase
-
sesquiterpene synthase, class I enzyme
aristolochene synthase
Q9UR08
-
aristolochene synthase
Q9UR08
sesquiterpene cyclase
aristolochene synthase
-
-
aristolochene synthase
Q03471
-
AS
-
-
-
-
cyclase, farnesyl pyrophosphate
-
-
-
-
EC 2.5.1.40
-
-
formerly
-
EC 4.1.99.7
-
-
formerly
-
farnesylpyrophosphate cyclase
-
-
-
-
FPP-carbocyclase
-
-
-
-
sesquiterpene cyclase
-
-
-
-
synthase, aristolochene
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
94185-89-4
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
expressed in
-
-
Manually annotated by BRENDA team
the crystal structure of aristolochene synthase, PDB code 1DGP, is used for an enzyme-substrate model constructed using CHARMM and insight II
UniProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2E,6E)-farnesyl diphosphate
(+)-5-epi-aristolochene + diphosphate
show the reaction diagram
Q03471
-
the steric bulk of residue 92 is central in binding of farnesyl diphosphate to the active site of aristolochene synthase in a quasi-cyclic conformation, thereby facilitating attack of C1 by the C10-C11 double bond to produce the cis-fused decalin S-germacrene A. Reduction of the size of the side chain of residue 92 leads to the production of the alicyclic sesquiterpenes (E)-beta- and (E,E)-alpha-farnesene. The relative amounts of linear products formed depend linearly on the size of the residues at position 92
-
?
(2E,6E)-farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
-
-
-
-
?
(2E,6E)-farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
-
-
major product (92%)
-
?
(2E,6E)-farnesyl diphosphate
(+)-5-epiaristolochene + diphosphate
show the reaction diagram
-
-
in addition to the major products (+)-5-epiaristolochene (78.9%), its DELTA1(10) isomer (-)-4-epieremophilene (6.2%), and (R)-germacrene A (3.7%), incubations of (2Z,6E)-farnesyl diphosphate with the enzyme lead to 22 additional sesquiterpenes (11% of total products), among the identified minor products are (-)-R-cedrene, an isomer of (-)-prezizaene and an acoradiene (together accounting for 2.5% of the total hydrocarbon fraction)
-
?
(2E,6E)-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
products are 91.5% aristolochene, 7.5% germacrene A, 1% valencene
-
?
(2E,6E)-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
reaction proceeds via germacrene A and eudesmane cation. The amount of germacrene A generated by mutant enzymes serves as a measure of the stabilization of eudesmane cation
wild-type, about 8% of germacrene A as by-product. His-tagged wild-type, about 4% of germacrene A
-
?
(2Z,6E)-farnesyl diphosphate
(+)-2-epiprezizaene + (-)-alpha-cedrene + (-)-beta-curcumene + alpha-acoradiene + 4-epi-alpha-acoradiene + alpha-bisabolol + epi-alpha-bisabolol + nerolidol + (2Z,6E)-farnesol + diphosphate
show the reaction diagram
-
-
(+)-2-epiprezizaene (44% yield), (-)-alpha-cedrene (21.5% yield), (-)-beta-curcumene (15.5% yield), alpha-acoradiene (3.9% yield), 4-epi-alpha-acoradiene (1.3% yield), alpha-bisabolol (1.8% yield), epi-alpha-bisabolol (1.8% yield), nerolidol (3.6% yield), (2Z,6E)-farnesol (6.7% yield)
-
?
14-fluoro (2E,6Z)-farnesyl diphosphate
14-fluorogermacrene A + diphosphate
show the reaction diagram
-
-
-
-
?
2-fluorofarnesyl diphosphate
2-fluorogermacrene A + diphosphate
show the reaction diagram
-
-
-
-
?
2-fluorofarnesyl diphosphate
2-fluorogermacrene A
show the reaction diagram
-
two products are identified in a 95/5 ratio by GS-MS
-
-
?
2-fluorofarnesyl-diphosphate
2-fluorogermacrene A
show the reaction diagram
-
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
Q03471
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-, Q9UR08
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
terpenoid biosynthesis
-
-
r
6-fluoro-(2E,6Z)-farnesyl diphosphate
6-fluorogermacrene A + diphosphate
show the reaction diagram
-
-
one of 3 major products
-
?
farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
-
-
-
-
-
farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
-
-
-
-
-
farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
-
biogenetic precursor of more than 300 different sesquiterpene hydrocarbon scaffolds in plants, bacteria and fungi
-
-
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
7.5% of the total amount of products are released from wild-type aristolochene synthase. The mutant enzyme Y92F releases significant amounts of germacrene A and also produces various amounts of a further five hydrocarbons of molecular weight 204, valencene, beta-(E)-farnesene, alpha-selinene, beta-selinene and selina-4,11-diene
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
cyclization of trans,trans-farnesyl diphosphate is proceeding with inversion of configuration at C-1 of farnesyl diphosphate
-
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
H8si is lost in the formation of the 9,10-double bond of aristolochene
-
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
metal-triggered carbocation formation initiates the cyclization cascade, which procedes through multiple complex intermediates to yield one exclusive structural stereochemical isomer of aristolochene
-
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
Q03471
the steric bulk of residue 92 is central in binding of farnesyl diphosphate to the active site of the enzyme in a quasi-cyclic conformation, thereby facilitating attack of C1 by the C10-C11 double bond to produce the cis-fused decalin S-germacrene A. The cyclization of farnesyl diphosphate to germacrene A in aristolochene synthase proceeds in a stepwise fashion through farnesyl cation
the mutant Y92A produces almost 80% of the alicyclic sesquiterpenes (E)-beta-farnesene and (E,E)-alpha-farnesene. The mutant also produces small amounts of additional hydrocarbons with a molecular weight of 204: alpha-selinene, beta-selinene, selina-4,11-diene, (E,Z)-alpha-farnesene, and beta-bisabolene
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
the enzyme appeears to be transcriptionally regulated
-
-
?
trans,trans-farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
-
-
(+)-aristolochene + minor amounts of (S)-(-)-germacrene A and (-)-valencene in a 94:4:2 ratio
-
?
trans,trans-farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
-
-
(+)-aristolochene is the only product
-
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate + germacrene
show the reaction diagram
-
-
wild-type enzyme produces 92% aristolochene, 8% germacrene and a small amount of valencene
-
?
farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
-
The universal sesquiterpene precursor farnesyl diphosphate (15-carbon isoprenoid) is cyclized in an Mg2-dependent reaction to form the bicyclic hydrocarbon aristolochene and a diphosphate anion coproduct
-
-
?
additional information
?
-
-
reaction is a a cyclisation cascade that leads to the generation of two 6-membered rings, three chiral centres, and two double bonds with high regio- and stereospecificity. Concurrent to diphosphate expulsion enzyme facilitates attack of C1 in farnesyl diphosphate by the C10, C11-double bond to produce germacryl cation. Proton loss from C12 leads to the production of (S)-germacrene A which is then postulated to undergo reprotonation of the C6, C7-double bond and a further cyclisation to form the bicyclic eudesmane cation. Successive 1,2-hydride shift and methyl migration followed by loss of H on C8 completes the generation of (+)-aristolochene
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
Q03471
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-, Q9UR08
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
terpenoid biosynthesis
-
-
r
farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
-
biogenetic precursor of more than 300 different sesquiterpene hydrocarbon scaffolds in plants, bacteria and fungi
-
-
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
the enzyme appeears to be transcriptionally regulated
-
-
?
farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
-
The universal sesquiterpene precursor farnesyl diphosphate (15-carbon isoprenoid) is cyclized in an Mg2-dependent reaction to form the bicyclic hydrocarbon aristolochene and a diphosphate anion coproduct
-
-
?
additional information
?
-
-
reaction is a a cyclisation cascade that leads to the generation of two 6-membered rings, three chiral centres, and two double bonds with high regio- and stereospecificity. Concurrent to diphosphate expulsion enzyme facilitates attack of C1 in farnesyl diphosphate by the C10, C11-double bond to produce germacryl cation. Proton loss from C12 leads to the production of (S)-germacrene A which is then postulated to undergo reprotonation of the C6, C7-double bond and a further cyclisation to form the bicyclic eudesmane cation. Successive 1,2-hydride shift and methyl migration followed by loss of H on C8 completes the generation of (+)-aristolochene
-
-
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Co2+
-
can substitute for Mg2+ over the concentration range of 0.16 -5.0 mM
Mg2+
-
required, maximal activity at 3 mM
Mg2+
-
divalent cation required, 5 mM Mg2+ preferred
Mg2+
-
required for enzyme reaction
Mg2+
-
enzyme utilizes a trinuclear magnesium cluster to trigger the departure of the diphosphate leaving group, thereby forming an allylic carbocation that typically reacts with one of the remaining sigma-bonds of the substrate
Mg2+
-
required
Mn2+
-
0.01 mM, can partially replace Mg2+, inhibition above
Mn2+
-
Mn2+ can replace Mg2+ at 0.1-0.2 mM
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
(4aS,7S)-1,4a-dimethyl-7-(prop-1-en-2-yl)-2,3,4,4a,5,6,7,8-octahydroquinolinium iodide
-
inhibitor mimics transition state associated with the cyclization of (-)-germacrene A to eudesmane cation, competitive
(4aS,7S)-4a-methyl-7-(prop-1-en-2-yl)-2,3,4,4a,5,6,7,8-octahydroquinolinium chloride
-
inhibitor mimics transition state associated with the cyclization of (-)-germacrene A to eudesmane cation, competitive
12,13-difluoro-farnesyl diphosphate
-
-
12,13-difluorofarnesyl diphosphate
-
-
12,13-difluorofarnesyl diphosphate
-
incubation of 12,13-difluorofarnesyl diphosphate with for 30 h does not generate any pentane-extractable products based on GC-MS analysis
3-phenylfarnesyl diphosphate
-
-
4-aza-eudesm-11-ene
-
transition state analogue, competitive
E-11-phenylfarnesyl diphosphate
-
-
farnesyl thiodiphosphate
-
-
Mn2+
-
above 0.01 mM, activation below
Mn2+
-
above 1.0 mM
Z-11-phenylfarnesyl diphosphate
-
-
additional information
-
no effect: phosphate up to concentrations of 5.0 mM
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.00013
-
(2E,6E)-farnesyl diphosphate
-
mutant W334H, pH not specified in the publication, temperature not specified in the publication
0.00028
-
(2E,6E)-farnesyl diphosphate
-
mutant W334F, pH not specified in the publication, temperature not specified in the publication
0.00053
-
(2E,6E)-farnesyl diphosphate
-
-
0.00053
-
(2E,6E)-farnesyl diphosphate
-
wild-type, pH not specified in the publication, temperature not specified in the publication
0.00064
-
(2E,6E)-farnesyl diphosphate
-
mutant V88A, pH not specified in the publication, temperature not specified in the publication
0.0007
-
(2E,6E)-farnesyl diphosphate
-
mutant V88F, pH not specified in the publication, temperature not specified in the publication
0.00075
-
(2E,6E)-farnesyl diphosphate
-
mutant W334L, pH not specified in the publication, temperature not specified in the publication
0.001
-
(2E,6E)-farnesyl diphosphate
-
mutant W334Y, pH not specified in the publication, temperature not specified in the publication
0.00175
-
(2E,6E)-farnesyl diphosphate
-
mutant T89A, pH not specified in the publication, temperature not specified in the publication
0.0023
-
(2E,6E)-farnesyl diphosphate
-
wild-type, pH 7.5, temperature not specified in the publication
0.005
-
(2E,6E)-farnesyl diphosphate
-
mutant T89F, pH not specified in the publication, temperature not specified in the publication
0.0502
-
(2E,6E)-farnesyl diphosphate
-
mutant Y92C, pH 7.5, temperature not specified in the publication
0.0834
-
(2E,6E)-farnesyl diphosphate
-
mutant Y92A, pH 7.5, temperature not specified in the publication
0.7
-
(2E,6E)-farnesyl diphosphate
-
mutant L108A, pH not specified in the publication, temperature not specified in the publication
0.99
-
(2E,6E)-farnesyl diphosphate
-
mutant L108S, pH not specified in the publication, temperature not specified in the publication
1.76
-
(2E,6E)-farnesyl diphosphate
-
mutant L108F, pH not specified in the publication, temperature not specified in the publication
2.03
-
(2E,6E)-farnesyl diphosphate
-
mutant L108V, pH not specified in the publication, temperature not specified in the publication
0.0143
-
(2Z,6E)-farnesyl diphosphate
-
in 200 mM Tris-HCl (pH 7.5), 40 mM MgCl2, at 22C
0.0008
-
farnesyl diphosphate
-
assay under normal conditions and in D2O, 20 mM Tris, 5 mM MgCl2, 5 mM 2-mercaptoethanol and 15% glycerol
0.0009
-
farnesyl diphosphate
-
mutant F178I
0.002
-
farnesyl diphosphate
-
mutant F178V
0.0023
-
farnesyl diphosphate
-
-
0.0047
-
farnesyl diphosphate
-
mutant F178W
0.0059
-
farnesyl diphosphate
-
mutant F178C
0.0087
-
farnesyl diphosphate
-
wild-type
0.0099
-
farnesyl diphosphate
-
mutant F178Y
0.0197
-
farnesyl diphosphate
-
mutant F112A
0.0463
-
farnesyl diphosphate
-
mutant F112A-178A
1.2e-05
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme Y92F
1.35e-05
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, native enzyme
1.48e-05
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, recombinant enzyme
0.00015
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme E119Q
0.00032
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme E119D
0.00052
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, recombinant enzyme
0.00055
-
trans,trans-farnesyl diphosphate
-
pH 7.5
0.0006
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, wild-type enzyme
0.0011
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme D116E
0.0013
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, wild-type enzyme
0.0014
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme E227D
0.0015
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme D116N
0.0023
-
trans,trans-farnesyl diphosphate
-
pH 7.5, 30C, recombinant wild-type enzyme
0.0027
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme D115E
0.0033
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme N244D
0.0034
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme E252D
0.0039
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme N219D
0.0051
-
trans,trans-farnesyl diphosphate
-
wild-type enzyme, mutant enzyme F178Y
0.0055
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme S248A
0.0101
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme E252Q
0.0112
-
trans,trans-farnesyl diphosphate
-
wild-type enzyme, mutant enzyme F178V
0.05027
-
trans,trans-farnesyl diphosphate
-
pH 7.5, mutant enzyme Y92C
0.0834
-
trans,trans-farnesyl diphosphate
-
pH 7.5, mutant enzyme Y92A
0.1887
-
trans,trans-farnesyl diphosphate
-
pH 7.5, 30C, mutant enzyme Y92F
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0003
-
(2E,6E)-farnesyl diphosphate
-
mutant W334H, pH not specified in the publication, temperature not specified in the publication
0.00049
-
(2E,6E)-farnesyl diphosphate
-
mutant Y92C, pH 7.5, temperature not specified in the publication
0.0008
-
(2E,6E)-farnesyl diphosphate
-
mutant V88F, pH not specified in the publication, temperature not specified in the publication
0.0009
-
(2E,6E)-farnesyl diphosphate
-
mutant L108A, pH not specified in the publication, temperature not specified in the publication
0.0011
-
(2E,6E)-farnesyl diphosphate
-
mutant T89F, pH not specified in the publication, temperature not specified in the publication
0.0014
-
(2E,6E)-farnesyl diphosphate
-
mutant Y92A, pH 7.5, temperature not specified in the publication
0.0025
-
(2E,6E)-farnesyl diphosphate
-
mutant V88A, pH not specified in the publication, temperature not specified in the publication
0.0027
-
(2E,6E)-farnesyl diphosphate
-
mutant W334L, pH not specified in the publication, temperature not specified in the publication
0.0036
-
(2E,6E)-farnesyl diphosphate
-
mutant L108F, pH not specified in the publication, temperature not specified in the publication
0.0057
-
(2E,6E)-farnesyl diphosphate
-
mutant L108V, pH not specified in the publication, temperature not specified in the publication
0.006
-
(2E,6E)-farnesyl diphosphate
-
mutant W334F, pH not specified in the publication, temperature not specified in the publication
0.0066
-
(2E,6E)-farnesyl diphosphate
-
mutant T89A, pH not specified in the publication, temperature not specified in the publication
0.007
-
(2E,6E)-farnesyl diphosphate
-
mutant W334Y, pH not specified in the publication, temperature not specified in the publication
0.0095
-
(2E,6E)-farnesyl diphosphate
-
mutant L108S, pH not specified in the publication, temperature not specified in the publication
0.03
-
(2E,6E)-farnesyl diphosphate
-
wild-type, pH 7.5, temperature not specified in the publication
0.084
-
(2E,6E)-farnesyl diphosphate
-
-
0.084
-
(2E,6E)-farnesyl diphosphate
-
wild-type, pH not specified in the publication, temperature not specified in the publication
0.095
-
(2Z,6E)-farnesyl diphosphate
-
in 200 mM Tris-HCl (pH 7.5), 40 mM MgCl2, at 22C
0.0014
-
farnesyl diphosphate
-
mutant F112A-178A
0.0023
-
farnesyl diphosphate
-
mutant F112A
0.0041
-
farnesyl diphosphate
-
mutant F178I
0.0063
-
farnesyl diphosphate
-
mutant F178V
0.0099
-
farnesyl diphosphate
-
mutant F178C
0.02
-
farnesyl diphosphate
-
assay under normal conditions and in D2O, 20 mM Tris, 5 mM MgCl2, 5 mM 2-mercaptoethanol and 15% glycerol
0.052
-
farnesyl diphosphate
-
mutant F178Y
0.079
-
farnesyl diphosphate
-
mutant F178W
0.55
-
farnesyl diphosphate
-
wild-type
1.4
-
farnesyl diphosphate
-
mutant F112A-178A
2.3
-
farnesyl diphosphate
-
mutant F112A
4.1
-
farnesyl diphosphate
-
mutant F178I
6.3
-
farnesyl diphosphate
-
mutant F178V
9.9
-
farnesyl diphosphate
-
mutant F178C
52
-
farnesyl diphosphate
-
mutant F178Y
79
-
farnesyl diphosphate
-
mutant F178W
550
-
farnesyl diphosphate
-
wild-type
2.1e-05
-
trans,trans-farnesyl diphosphate
-
wild-type enzyme, mutant enzyme F178V
7.6e-05
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme N219D
0.00012
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme N244D
0.00022
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme E252Q
0.0004
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme Y92F
0.000491
-
trans,trans-farnesyl diphosphate
-
pH 7.5, mutant enzyme Y92C
0.001
-
trans,trans-farnesyl diphosphate
-
wild-type enzyme, mutant enzyme F178Y
0.0013
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme D116E; pH 8.0, 30C, mutant enzyme S248A
0.00137
-
trans,trans-farnesyl diphosphate
-
pH 7.5, mutant enzyme Y92A
0.0016
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme E227D
0.0022
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme D116N; pH 8.0, 30C, mutant enzyme E252D
0.0023
-
trans,trans-farnesyl diphosphate
-
pH 7.5, 30C, mutant enzyme Y92F
0.0048
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme E119Q
0.0097
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme E119D
0.014
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, native enzyme
0.015
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, recombinant enzyme
0.0162
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, mutant enzyme D115E
0.0173
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, wild-type enzyme
0.03
-
trans,trans-farnesyl diphosphate
-
pH 7.5, 30C, recombinant wild-type enzyme
0.043
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, recombinant enzyme
0.043
-
trans,trans-farnesyl diphosphate
-
pH 8.0, 30C, wild-type enzyme
kcat/KM VALUE [1/mMs-1]
kcat/KM VALUE [1/mMs-1] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0011
-
(2E,6E)-farnesyl diphosphate
-
mutant V88F, pH not specified in the publication, temperature not specified in the publication
263972
0.0013
-
(2E,6E)-farnesyl diphosphate
-
mutant L108A, pH not specified in the publication, temperature not specified in the publication
263972
0.0021
-
(2E,6E)-farnesyl diphosphate
-
mutant L108F, pH not specified in the publication, temperature not specified in the publication
263972
0.0022
-
(2E,6E)-farnesyl diphosphate
-
mutant T89F, pH not specified in the publication, temperature not specified in the publication
263972
0.0028
-
(2E,6E)-farnesyl diphosphate
-
mutant L108V, pH not specified in the publication, temperature not specified in the publication
263972
0.0038
-
(2E,6E)-farnesyl diphosphate
-
mutant T89A, pH not specified in the publication, temperature not specified in the publication
263972
0.0039
-
(2E,6E)-farnesyl diphosphate
-
mutant V88A, pH not specified in the publication, temperature not specified in the publication
263972
0.0096
-
(2E,6E)-farnesyl diphosphate
-
mutant L108S, pH not specified in the publication, temperature not specified in the publication
263972
0.01
-
(2E,6E)-farnesyl diphosphate
-
mutant Y92C, pH 7.5, temperature not specified in the publication
263972
0.016
-
(2E,6E)-farnesyl diphosphate
-
mutant Y92A, pH 7.5, temperature not specified in the publication
263972
0.158
-
(2E,6E)-farnesyl diphosphate
-
wild-type, pH not specified in the publication, temperature not specified in the publication
263972
2.56
-
(2E,6E)-farnesyl diphosphate
-
mutant W334H, pH not specified in the publication, temperature not specified in the publication
263972
3.56
-
(2E,6E)-farnesyl diphosphate
-
mutant W334L, pH not specified in the publication, temperature not specified in the publication
263972
5
-
(2E,6E)-farnesyl diphosphate
-
wild-type protein
263972
5
-
(2E,6E)-farnesyl diphosphate
-
-
263972
6.95
-
(2E,6E)-farnesyl diphosphate
-
mutant W334Y, pH not specified in the publication, temperature not specified in the publication
263972
11.8
-
(2E,6E)-farnesyl diphosphate
-
A274T/V372I/Y406L/V516I mutant protein
263972
13.04
-
(2E,6E)-farnesyl diphosphate
-
wild-type, pH 7.5, temperature not specified in the publication
263972
22.6
-
(2E,6E)-farnesyl diphosphate
-
mutant W334F, pH not specified in the publication, temperature not specified in the publication
263972
118
-
(2E,6E)-farnesyl diphosphate
-
-
263972
158.5
-
(2E,6E)-farnesyl diphosphate
-
wild-type, pH not specified in the publication, temperature not specified in the publication
263972
0.0068
-
(2Z,6E)-farnesyl diphosphate
-
in 200 mM Tris-HCl (pH 7.5), 40 mM MgCl2, at 22C
230497
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.219
-
(4aS,7S)-1,4a-dimethyl-7-(prop-1-en-2-yl)-2,3,4,4a,5,6,7,8-octahydroquinolinium iodide
-
pH not specified in the publication, temperature not specified in the publication
0.0284
-
(4aS,7S)-4a-methyl-7-(prop-1-en-2-yl)-2,3,4,4a,5,6,7,8-octahydroquinolinium chloride
-
pH not specified in the publication, temperature not specified in the publication
0.0008
-
12,13-difluorofarnesyl diphosphate
-
-
0.0012
-
3-phenylfarnesyl diphosphate
-
-
0.00024
-
4-aza-eudesm-11-ene, ammonium salt
-
in the presence of 0.25 mM diphosphate
0.00035
-
4-aza-eudesm-11-ene, ammonium salt
-
-
0.0008
-
E-11-phenylfarnesyl diphosphate
-
-
0.01
-
farnesyl thiodiphosphate
-
-
0.0012
-
Z-11-phenylfarnesyl diphosphate
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.0236
-
-
native enzyme
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.5
-
-
preparative scale incubation, 48 h
7.5
-
-
activity assay
7.5
-
-
assay at
7.6
-
-
assay at
8
-
-
recombinant enzyme, HEPES buffer
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
preparative scale incubation, 48 h
25
-
-
activity assay
25
-
-
assay at
30
-
-
activity assay
30
-
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
48000
-
-
gel filtration
70000
-
-
dimer, native gel analysis
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 39200, calculation from nucleotide sequence
?
-
x * 36480, calculation from nucleotide sequence; x * 39000, SDS-PAGE
dimer
-
in solution
monomer
-
1 * 37000, SDS-PAGE
monomer
-
39000, calculated
tetramer
-
crystal structure
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
crystallization by the hanging drop, vapor diffusion method at 4C, the crystal structure determined from crystals soaked with farnesyl diphosphate reveals the binding of intact farnesyl diphosphate to monomers A-C, and the binding of diphosphate anion and Mg2+ to monomer D. The structure of the complex with 2-fluorofarnesyl diphosphate reveals 2-fluorofarnesyl diphosphate binding to all subunits of the tetramer, with Mg2+B accompanying the binding of this analogue only in monomer D. The structure of the complex with 12,13-difluorofarnesyl diphosphate reveals the binding of intact 12,13-difluorofarnesyl diphosphate to monomers A-C in the open conformation and the binding of diphosphate anion, Mg2+B, and Mg2+C to monomer D in a predominantly closed conformation
-
the structure of recombinant aristolochene synthase at a resolution of 2.2 A and its complex with diphosphate and three Mg2+ ions at 2.15 A is reported
-
2.5-A resolution crystal structure of recombinant enzyme, hanging drop method
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
native and recombinant enzyme
-
using DE52 anion exchange resin, a methyl HIC, a Sephadex G-25, and a High Q column
-
mutant enzyme Y92C and Y92A
-
Proteins are extracted from the inclusion bodies and purified following established protocols, each enzyme is pure as judged by SDS-gel electrophoresis
-
recombinant enzyme
-
wild-type and mutant enzymes
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
high-level expression in Escherichia coli
-
into the pET11 vector for expression in Escherichia coli BL21DE3pLysS cells
-
recombinant aristolochene synthase is expressed in Escherichia coli BL21(DE3)pLysS
-
expressed in Escherichia coli BL21(DE3)
-
expression of AS and AS-F112A in Escherichia coli
-
expression of the fusion protein proteinA/aristolochene synthase in Escherichia coli
-
for expression in Escherichia coli BL21DE3 cells
-
mutant enzymes Y92C and Y92A
-
wild-type and mutant enzyme Y92F, expression in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
E227D
-
kcat/KM is 1182fold higher than wild-type value, in contrast to wild-type enzyme that exclusively produces aristolochene from trans,trans-farnesyl diphosphate, the mutant enzyme produces 26% aristolochene and 74% germacrene A
E227Q
-
inactive mutant enzyme
N219D
-
kcat/KM is 6667fold higher than wild-type value, in contrast to wild-type enzyme that exclusively produces aristolochene from trans,trans-farnesyl diphosphate, the mutant enzyme produces 44% aristolochene and 56% germacrene A
D115E
-
kcat/KM is 12fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 75:6:119
D115N
-
inactive mutant enzyme
D116E
-
kcat/KM is 60fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 62:3:35
D116N
-
kcat/KM is 48fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 63:2:35
E119D
-
kcat/KM is 2.4fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 94:2:4
E119Q
-
kcat/KM is 2.3fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 84:2:14
E252D
-
kcat/KM is 111fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 19:0:81
E252Q
-
mutant enzyme produces only (-)-germacrene A
F112A
-
residue 112 contributes to the stabilisation of the transition state following farnesyl cation
F112A
-
expression of AS-F112A in Escherichia coli
F112A/F178A
-
mutant is constructed to confirm the proposed roles of both F178 and F112
F178C
-
mutant is constructed to distinguish between the importance of size and aromaticity residue 178
F178I
-
mutant is constructed to distinguish between the importance of size and aromaticity residue 178
F178V
-
wild-type enzyme produces 92% aristolochene, 8% germacrene and a small amount of valencene. Mutant enzyme produces 10.8% aristolochene, 54.1% germacrene, 5.2% valencene, 5.7% alpha-selinene, 9.1% beta-selinine, 9.2% (E)-beta-farnesene and 2.7% (E,E)-alpha-farnesene. kcat is 1429fold lower than wild-type value, Km-value is 4.9fold higher than wild-type value
F178V
-
mutation leads to the accumulation of the intermediate germacrene A, the production of selinenes and the linear alpha- and beta-farnesene
F178W
-
mutant is constructed to distinguish between the importance of size and aromaticity residue 178
F178Y
-
wild-type enzyme produces 92% aristolochene, 8% germacrene and a small amount of valencene. Mutant enzyme produces 86.4% aristolochene, 10.7% germacrene, and 2.7% valencene. kcat is 30fold lower than wild-type value, Km-value is 2.2fold higher than wild-type value
F178Y
-
mutant is constructed to distinguish between the importance of size and aromaticity residue 178
L108A
-
products are 6.8% aristolochene, 13.9% germacrene A, 26.9% (E)-beta-farnesene, 48.2%(E,E)-alpha-farnesene and 4.1% (E,Z)-alpha-farnesene
L108F
-
products are 74.1% aristolochene, 13.8% germacrene A, 12% valencene
L108S
-
products are 9.4% aristolochene, 14.6% germacrene A, 21.5% (E)-beta-farnesene, 49.1% (E,E)-alpha-farnesene and 5.3% (E,Z)-alpha-farnesene
N244D
-
kcat/KM is 1978fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 19:0:81
N244L
-
inactive mutant enzyme
S248A
-
kcat/KM is 300fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 21:0:79
S248A/E252D
-
inactive mutant enzyme
T89A
-
products are 93.4% aristolochene, 4.4% germacrene A, 2.2% valencene
T89F
-
products are 67.6% aristolochene, 27.2% germacrene A, 5.2% valencene
V88A
-
products are 86.2% aristolochene, 11.6% germacrene A, 2.2% valencene
V88F
-
products are 18.4% aristolochene, 57.8% germacrene A, 23.8% valencene
W334F
-
ratio of products: 82% aristolochene, 18% germacrene A
W334H
-
ratio of products: 10% aristolochene, 90% germacrene A
W334L
-
ratio of products: 2% aristolochene, 98% germacrene A
W334Y
-
ratio of products: 62% aristolochene, 38% germacrene A
Y92A
-
reduction of the size of the side chain of residue 92 leads to the production of the alicyclic sesquiterpenes (E)-beta- and (E,E)-alpha-farnesene. The relative amounts of linear products formed depend linearly on the size of the residues at position 92. ASY92A produces almost 80% of alicyclic sesquiterpenes and no aristolochene; turnover number is approximately 2 orders of magnitude lower than the value observed for the wild-type enzyme,the mutant enzyme produces almost 80% of the alicyclic sesquiterpenes (E)-beta-farnesene and (E,E)-alpha-farnesene. The mutant also produces small amounts of additional hydrocarbons with a molecular weight of 204: alpha-selinene, beta-selinene, selina-4,11-diene, (E,Z)-alpha-farnesene, and beta-bisabolene. Km-value for trans, trans-farnesyl diphosphate is 0.0834 mM compared to 0.0023 mM for the wild-type enzyme
Y92C
-
reduction of the size of the side chain of residue 92 leads to the production of the alicyclic sesquiterpenes (E)-beta- and (E,E)-alpha-farnesene. Mutant ASY92C still produces about 6.8% of aristolochene; turnover number is approximately 2 orders of magnitude lower than the value observed for the wild-type enzyme. Km-value for trans, trans-farnesyl diphosphate is 0.05027 mM compared to 0.0023 mM for the wild-type enzyme
Y92F
-
the mutant enzyme is approximately 0.1% as active as the nonmutated recombinant enzyme, the mutant releases significant amounts of germacrene A and also produces various amounts of a further five hydrocarbons of molecular weight 204, valencene, beta-(E)-farnesene, alpha-selinene, beta-selinene and selina-4,11-diene. The CD spectrum of the mutant enzyme is very similar to that of the wild-type enzyme
Y92F
-
reduction of the size of the side chain of residue 92 leads to the production of the alicyclic sesquiterpenes (E)-beta- and (E,E)-alpha-farnesene
Y92F
-
100fold reduction in kcat, 50fold decrease in KM, resulting in 2fold decrease in kcat/Km. The mutant enzyme produces (+)-aristolochene as 81% of the product, 7% (-)-valencene and 12% (S)-(-)-germacrene A
Y92V
-
the mutant produces the alicyclic beta-(E)-farnesene as the major product
Y92V
-
reduction of the size of the side chain of residue 92 leads to the production of the alicyclic sesquiterpenes (E)-beta- and (E,E)-alpha-farnesene
L108V
-
products are 88.3% aristolochene, 8% germacrene A, 1% valencene, and 2.3% (E,Z)-alpha-farnesene
additional information
-
the replacement of Trp 334 with para-substituted phenylalanines of increasing electron-withdrawing properties leads to a progressive accumulation of germacrene A that shows a good correlation with the interaction energies of simple cations such as Na+ with substituted benzenes. Evidence for the stabilizing role played by Trp334 in aristolochene synthase catalysis for the energetically demanding transformation of germacrene A to eudesmane cation. Replacement of tryptophan by para-substituted phenylalanines with strong electron-withdrawing substituents has only minor effects on the KM values of the reaction but leads to approximately 30fold decreases of kcat relative to mutant W334F. Noncanonical substitutions lead to the following ratios of products: W334naphthyl 78% aristolochene, 22% germacrene A, W334-p-chlorophenylalanine or W334-p-fluorophenylalanine 57% aristolochene, 43% germacrene A, W334-p-trifluoromethylphenylalanine 31% aristolochene, 69% germacrene A, W334-p-nitrophenylalanine 23% aristolochene, 77% germacrene A
Renatured/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
extracted from inclusion bodies
-
protein is extracted from inclusion bodies
-