| Terpenoids are the major components of natural metabolites from plants. So far, morethan55,000classes of terpenoids have been isolated and identified from nature, and the largeamount and diverse structures of terpenoids are both resulted from the activities of terpenesynthases. As a result of the finding of large quantities of valuable terpenoids, it’s moreimportant to study the terpene synthases. The research on terpene synthases would not onlyhelp us understand their catalytic mechanism and the relationship between structure andfunction in terms of theory, but also contribute definitely to the development of new drugsfrom terpenoids in practical application. The most interesting part in the study of terpenoidsis how creatures can specifically produce such large amount of terpenoids, and catalyticmechanism of terpene synthases, especially catalytic specificity, is a key to this question.We have done the following works on exploring the catalytic mechanism of SesquiterpeneSynthses:1. To study the effect of C-terminus on the enzyme activity, we did a series of truncation(6,7,8,9,10,29amino acids) to C-terminus of Amorpha-4,11-diene synthase of Artemisiaannua(ADS). We found that enzyme activity was a little decreased by the truncation of6amino acids (C6), activity was almost lost by C7, activities were extraordinarily weak byC8-C10, and activities were totally lost by C14and C29. The results showed that, althoughC-terminus is not involved in the formation of active center, its structure is very important forthe maintaining of the enzyme activity.2. To study the effect of helix G kink on ADS activity, we introduced both singlemutation and joint mutation including G400, G401, A402, and L405. The product specificitiesof enzyme were changed by all the mutations except A402. G401A mutation led to increasedratios of γ-humulene and amorpha-4,7(11)-diene, which indicated that G401might beassociated with deprotonation of final products. G400A mutation only increased the ratio ofγ-humulene. Double mutation of G401A and G400A induced further increase ofamorpha-4,7(11)-diene and γ-humulene compared to single mutation of G401A, showing codominance of G400A and G401A. Single mutation of T399L caused increased ratio ofamorpha-4,7(11)-diene, and double mutation of T399L and G401A resulted in elimination ofγ-humlene in addition to increased amorpha-4,7(11)-diene, which implied that T399Linhibits the production of γ-humulene and indicated epistatic effect of T399L upon G401A.Shortened side chain from L405A mutation would lead to enlarged pocket and thus result infailure of the second cyclization, but has no effect on the first cyclization. Shortened sidechain also makes it easy for water molecule to enter active center, leading to the production ofsesquiterpenoid alchol.3. To study the effect of RxR motif on enzyme activity, we induced point mutation toRxR motif and the related D300. We found that in conserved motif, single mutation of R262Kchanged the major product of enzymatic reaction to be (R,E)-Nerolidol, while R262L singlemutation inactivated the enzyme and R264K single mutation had not effect on the ratio ofproducts. The results indicated that the first arginine in the conserved motif is very critical forthe maintaining of hydrophobic environment of active center. The result from D300mutationshowed a pretty necessary function of this amino acid for the activity of the enzyme.Combined to the result from R262, it is estimated that the non-bonded interaction betweenR262and D300plays an important role for the maintaining of enzyme activity. |
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