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 hide
4.2.3.9
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RECOMMENDED NAME
GeneOntology No.
aristolochene synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(2E,6E)-farnesyl diphosphate = aristolochene + diphosphate
show the reaction diagram
further cyclization and methyl transfer converts the intermediate into aristolochene
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cyclization
internal cyclization
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
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Metabolic pathways
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Sesquiterpenoid and triterpenoid biosynthesis
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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.
CAS REGISTRY NUMBER
COMMENTARY hide
94185-89-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
expressed in
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2E,6E)-farnesyl diphosphate
(+)-5-epi-aristolochene + diphosphate
show the reaction diagram
(2E,6E)-farnesyl diphosphate
(+)-5-epiaristolochene + diphosphate
show the reaction diagram
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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)
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?
(2E,6E)-farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
(2E,6E)-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
(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
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(+)-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)
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?
14-fluoro (2E,6Z)-farnesyl diphosphate
14-fluorogermacrene A + diphosphate
show the reaction diagram
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?
2-fluorofarnesyl diphosphate
2-fluorogermacrene A
show the reaction diagram
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two products are identified in a 95/5 ratio by GS-MS
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?
2-fluorofarnesyl diphosphate
2-fluorogermacrene A + diphosphate
show the reaction diagram
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?
2-fluorofarnesyl-diphosphate
2-fluorogermacrene A
show the reaction diagram
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?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
6-fluoro-(2E,6Z)-farnesyl diphosphate
6-fluorogermacrene A + diphosphate
show the reaction diagram
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one of 3 major products
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?
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 + germacrene
show the reaction diagram
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wild-type enzyme produces 92% aristolochene, 8% germacrene and a small amount of valencene
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?
additional information
?
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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
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
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the enzyme appeears to be transcriptionally regulated
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?
additional information
?
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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
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
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can substitute for Mg2+ over the concentration range of 0.16 -5.0 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(4aS,7S)-1,4a-dimethyl-7-(prop-1-en-2-yl)-2,3,4,4a,5,6,7,8-octahydroquinolinium iodide
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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
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inhibitor mimics transition state associated with the cyclization of (-)-germacrene A to eudesmane cation, competitive
12,13-difluoro-farnesyl diphosphate
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12,13-difluorofarnesyl diphosphate
3-phenylfarnesyl diphosphate
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4-aza-eudesm-11-ene
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transition state analogue, competitive
E-11-phenylfarnesyl diphosphate
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farnesyl thiodiphosphate
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Z-11-phenylfarnesyl diphosphate
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additional information
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no effect: phosphate up to concentrations of 5.0 mM
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00013 - 2.03
(2E,6E)-farnesyl diphosphate
0.0143
(2Z,6E)-farnesyl diphosphate
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in 200 mM Tris-HCl (pH 7.5), 40 mM MgCl2, at 22C
0.0008 - 0.0463
farnesyl diphosphate
0.000012 - 0.1887
trans,trans-farnesyl diphosphate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0003 - 0.084
(2E,6E)-farnesyl diphosphate
0.095
(2Z,6E)-farnesyl diphosphate
Nicotiana tabacum
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in 200 mM Tris-HCl (pH 7.5), 40 mM MgCl2, at 22C
0.0014 - 550
farnesyl diphosphate
0.000021 - 0.043
trans,trans-farnesyl diphosphate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0011 - 158.5
(2E,6E)-farnesyl diphosphate
0.0068
(2Z,6E)-farnesyl diphosphate
Nicotiana tabacum
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in 200 mM Tris-HCl (pH 7.5), 40 mM MgCl2, at 22C
1810
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
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
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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
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pH not specified in the publication, temperature not specified in the publication
0.0008
12,13-difluorofarnesyl diphosphate
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0.0012
3-phenylfarnesyl diphosphate
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0.00024 - 0.00035
4-aza-eudesm-11-ene, ammonium salt
0.0008
E-11-phenylfarnesyl diphosphate
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0.01
farnesyl thiodiphosphate
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0.0012
Z-11-phenylfarnesyl diphosphate
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0236
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native enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.6
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assay at
8
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recombinant enzyme, HEPES buffer
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
48000
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gel filtration
70000
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dimer, native gel analysis
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
tetramer
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crystal structure
Crystallization/COMMENTARY
ORGANISM
UNIPROT
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
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molecular dynamics simulations based on structure PDB entry 2OA6. The substrate farnesyl diphosphate binds first, followed by three magnesium ions in sequence, and, after reaction, the release of aristolochene and two magnesium ions followed by the final magnesium ion and diphosphate. Binding of farnesyl diphosphate leads to an increased level of sampling of open conformations, allowing the first two magnesium ions to bind. The closed enzyme conformation is maintained with a diphosphate moiety and two magnesium ions bound. The open-to-closed transition reduces flexibility around the active site entrance, partly through a lid closing over it
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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
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X-ray crystal structure of aristolochene synthase complexed with three Mg2+ ions and the unreactive substrate analogue farnesyl-S-thiolodiphosphate, showing that the substrate diphosphate group is anchored by metal coordination and hydrogen bond interactions. The binding conformation of farnesyl-S-thiolodiphosphate directly mimics that expected for productively bound farnesyl diphosphate, with the exception of the precise alignment of the C-S bond with regard to the C10-C11 pi system that would be required for C1-C10 bond formation in the first step of catalysis. Crystal structures of aristolochene synthase complexed with Mg2+3-diphosphate and ammonium or iminium analogues of bicyclic carbocation intermediates proposed for the natural cyclization cascade show various binding orientations for these bicyclic analogues, which appear to be driven by favorable electrostatic interactions between the positively charged ammonium group of the analogue and the negatively charged diphosphate anion. The active site is sufficiently flexible to accommodate analogues with partially or completely incorrect stereochemistry
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2.5-A resolution crystal structure of recombinant enzyme, hanging drop method
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
mutant enzyme Y92C and Y92A
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native and recombinant enzyme
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Proteins are extracted from the inclusion bodies and purified following established protocols, each enzyme is pure as judged by SDS-gel electrophoresis
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recombinant enzyme
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using DE52 anion exchange resin, a methyl HIC, a Sephadex G-25, and a High Q column
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wild-type and mutant enzymes
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3)
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expression of AS and AS-F112A in Escherichia coli
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expression of the fusion protein proteinA/aristolochene synthase in Escherichia coli
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for expression in Escherichia coli BL21DE3 cells
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high-level expression in Escherichia coli
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into the pET11 vector for expression in Escherichia coli BL21DE3pLysS cells
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mutant enzymes Y92C and Y92A
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recombinant aristolochene synthase is expressed in Escherichia coli BL21(DE3)pLysS
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wild-type and mutant enzyme Y92F, expression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E227D
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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
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inactive mutant enzyme
N219D
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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
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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
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inactive mutant enzyme
D116E
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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
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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
D203L
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approximately 150fold decrease in kcat/KM
E119D
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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
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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
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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
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mutant enzyme produces only (-)-germacrene A
F112A/F178A
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mutant is constructed to confirm the proposed roles of both F178 and F112
F178C
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mutant is constructed to distinguish between the importance of size and aromaticity residue 178
F178I
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mutant is constructed to distinguish between the importance of size and aromaticity residue 178
F178W
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mutant is constructed to distinguish between the importance of size and aromaticity residue 178
K251Q
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20fold reduction in catalytic efficiency
K251R
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6fold reduction in catalytic efficiency
L108A
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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
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products are 74.1% aristolochene, 13.8% germacrene A, 12% valencene
L108S
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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
L108V
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products are 88.3% aristolochene, 8% germacrene A, 1% valencene, and 2.3% (E,Z)-alpha-farnesene
N244D
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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
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inactive mutant enzyme
R200E
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inactive
R200K
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approximately 300fold decrease in kcat/KM, mutant produces significantly increased amounts of germacrene A
R200Q
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inactive
R340K
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approximately 300fold decrease in kcat/KM, mutant produces significantly increased amounts of germacrene A
R340M
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inactive
S248A
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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
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inactive mutant enzyme
T89A
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products are 93.4% aristolochene, 4.4% germacrene A, 2.2% valencene
T89F
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products are 67.6% aristolochene, 27.2% germacrene A, 5.2% valencene
V88A
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products are 86.2% aristolochene, 11.6% germacrene A, 2.2% valencene
V88F
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products are 18.4% aristolochene, 57.8% germacrene A, 23.8% valencene
W334F
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ratio of products: 82% aristolochene, 18% germacrene A
W334H
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ratio of products: 10% aristolochene, 90% germacrene A
W334L
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ratio of products: 2% aristolochene, 98% germacrene A
W334Y
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ratio of products: 62% aristolochene, 38% germacrene A
Y341F
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10fold reduction in catalytic efficiency
Y92A
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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
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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
additional information
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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
LITERATURE
extracted from inclusion bodies
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protein is extracted from inclusion bodies
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