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4.2.3.9: aristolochene synthase

This is an abbreviated version!
For detailed information about aristolochene synthase, go to the full flat file.

Word Map on EC 4.2.3.9

Reaction

(2E,6E)-farnesyl diphosphate
=
aristolochene
 +
diphosphate

Synonyms

5-epi-aristolochene synthase, Ari1, aristolochene synthase, AS, ATAS, cyclase, farnesyl pyrophosphate, EAS, EC 2.5.1.40, EC 4.1.99.7, farnesylpyrophosphate cyclase, FPP-carbocyclase, sesquiterpene cyclase, synthase, aristolochene, TEAS

ECTree

     4 Lyases
         4.2 Carbon-oxygen lyases
             4.2.3 Acting on phosphates
                4.2.3.9 aristolochene synthase

Engineering

Engineering on EC 4.2.3.9 - aristolochene synthase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
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
N299A
the mutation has decreasing effects on catalysis but significant effects on sesquiterpene product distributions of 50% germacrene A and 50% aristolochene
N299A/S303A
the decreasing have modest effects on catalysis but significant effects on sesquiterpene product distributions with germacrene A as main product and (E)-nerolidol and aristolochene as by-products
N299L
the mutation has decreasing effects on sesquiterpene product distributions with germacrene A and aristolochene as main product and (E)-nerolidol as by-product
Q151E
the mutation has decreasing effects on catalysis but significant effects on sesquiterpene product distributions with aristolochene and germacrene A as main products, and (E)-nerolidol and (E,E)-farnesol as by-products
Q151H
the mutation has decreasing effects on catalysis but significant effects on sesquiterpene product distributions with germacrene A and (E,E)-farnesol as main products and (E)-nerolidol and aristolochene as by-products
S303A
the mutation has increasing effects on catalysis and significant effects on sesquiterpene product distributions with germacrene A and (E)-nerolidol as by-products
S303D
the mutation has decreasing effects on catalysis but significant effects on sesquiterpene product distributions with germacrene A and (E,E)-farnesol as main products and (E)-nerolidol and aristolochene as by-products
S303H
the mutation has decreasing effects on catalysis but significant effects on sesquiterpene product distributions with germacrene A as main product and (E,E)-farnesol, (E)-nerolidol and aristolochene as by-products
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
D203L
approximately 150fold decrease in kcat/KM
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
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
F178W
-
mutant is constructed to distinguish between the importance of size and aromaticity residue 178
F178Y
K251Q
20fold reduction in catalytic efficiency
K251R
6fold reduction in catalytic efficiency
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
L108V
products are 88.3% aristolochene, 8% germacrene A, 1% valencene, and 2.3% (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
R200K
approximately 300fold decrease in kcat/KM, mutant produces significantly increased amounts of germacrene A
R340K
approximately 300fold decrease in kcat/KM, mutant produces significantly increased amounts of germacrene A
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
Y341F
10fold reduction in catalytic efficiency
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