4.2.3.9 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 665301 4.2.3.9 D115N inactive mutant enzyme 665301 4.2.3.9 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 665301 4.2.3.9 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 665301 4.2.3.9 D203L approximately 150fold decrease in kcat/KM 729580 4.2.3.9 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 665301 4.2.3.9 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 665301 4.2.3.9 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 665301 4.2.3.9 E227Q inactive mutant enzyme 665301 4.2.3.9 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 665301 4.2.3.9 E252Q mutant enzyme produces only (-)-germacrene A 665301 4.2.3.9 F112A expression of AS-F112A in Escherichia coli 694515 4.2.3.9 F112A residue 112 contributes to the stabilisation of the transition state following farnesyl cation 682223 4.2.3.9 F112A/F178A mutant is constructed to confirm the proposed roles of both F178 and F112 682223 4.2.3.9 F178C mutant is constructed to distinguish between the importance of size and aromaticity residue 178 682223 4.2.3.9 F178I mutant is constructed to distinguish between the importance of size and aromaticity residue 178 682223 4.2.3.9 F178V mutation leads to the accumulation of the intermediate germacrene A, the production of selinenes and the linear alpha- and beta-farnesene 682223 4.2.3.9 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 664584 4.2.3.9 F178W mutant is constructed to distinguish between the importance of size and aromaticity residue 178 682223 4.2.3.9 F178Y mutant is constructed to distinguish between the importance of size and aromaticity residue 178 682223 4.2.3.9 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 664584 4.2.3.9 K251Q 20fold reduction in catalytic efficiency 729580 4.2.3.9 K251R 6fold reduction in catalytic efficiency 729580 4.2.3.9 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 716415 4.2.3.9 L108F products are 74.1% aristolochene, 13.8% germacrene A, 12% valencene 716415 4.2.3.9 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 716415 4.2.3.9 L108V products are 88.3% aristolochene, 8% germacrene A, 1% valencene, and 2.3% (E,Z)-alpha-farnesene 716415 4.2.3.9 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 715288 4.2.3.9 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 665301 4.2.3.9 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 665301 4.2.3.9 N244L inactive mutant enzyme 665301 4.2.3.9 N299A the mutation has decreasing effects on catalysis but significant effects on sesquiterpene product distributions of 50% germacrene A and 50% aristolochene 747103 4.2.3.9 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 747103 4.2.3.9 N299L the mutation has decreasing effects on sesquiterpene product distributions with germacrene A and aristolochene as main product and (E)-nerolidol as by-product 747103 4.2.3.9 N299V inactive 747103 4.2.3.9 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 747103 4.2.3.9 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 747103 4.2.3.9 R200E inactive 729580 4.2.3.9 R200K approximately 300fold decrease in kcat/KM, mutant produces significantly increased amounts of germacrene A 729580 4.2.3.9 R200Q inactive 729580 4.2.3.9 R340K approximately 300fold decrease in kcat/KM, mutant produces significantly increased amounts of germacrene A 729580 4.2.3.9 R340M inactive 729580 4.2.3.9 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 665301 4.2.3.9 S248A/E252D inactive mutant enzyme 665301 4.2.3.9 S303A the mutation has increasing effects on catalysis and significant effects on sesquiterpene product distributions with germacrene A and (E)-nerolidol as by-products 747103 4.2.3.9 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 747103 4.2.3.9 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 747103 4.2.3.9 T89A products are 93.4% aristolochene, 4.4% germacrene A, 2.2% valencene 716415 4.2.3.9 T89F products are 67.6% aristolochene, 27.2% germacrene A, 5.2% valencene 716415 4.2.3.9 V88A products are 86.2% aristolochene, 11.6% germacrene A, 2.2% valencene 716415 4.2.3.9 V88F products are 18.4% aristolochene, 57.8% germacrene A, 23.8% valencene 716415 4.2.3.9 W334F ratio of products: 82% aristolochene, 18% germacrene A 715288 4.2.3.9 W334H ratio of products: 10% aristolochene, 90% germacrene A 715288 4.2.3.9 W334L ratio of products: 2% aristolochene, 98% germacrene A 715288 4.2.3.9 W334Y ratio of products: 62% aristolochene, 38% germacrene A 715288 4.2.3.9 Y341F 10fold reduction in catalytic efficiency 729580 4.2.3.9 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 644243 4.2.3.9 Y92A 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 644243 4.2.3.9 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 644243 4.2.3.9 Y92C 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 644243 4.2.3.9 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 665301 4.2.3.9 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 644243 4.2.3.9 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 644242 4.2.3.9 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 644243 4.2.3.9 Y92V the mutant produces the alicyclic beta-(E)-farnesene as the major product 644241