Research motive

The oxidosqualene cyclases family and their sterol products have fascinated scientists for more than half of a century. Not only biochemists, but also organic chemists and physical chemists, even pharmaceutical scientists and doctors are conducting research topic on this family of molecules. The goals of these studies are to understand the enzyme mechanisms, to make artificial enzymes to synthesize specific products, to find inhibitors to against bacteria or fungi, and to produce drugs to lower cholesterol levels or to kill cancer cells for various therapies.

The mechanism of the oxidosqualene cyclases activity involves cyclization and rearrangement. In fact, only one enzyme and one step is needed during the entire process of this amazing organic reaction. In addition, a few amino acid alterations could make a large stereochemical change on the product profiles. Currently, however, it is still troublesome to synthesize a compound when the processes are complicated, and involve a mass of chemicals. Studies on mechanisms in this area of chemistry could lead us to understand the relationship between the active-site amino acids and the appearances of products. In the future, synthesizing a complicated compound by using a few artificial enzymes is a goal worth achieving.

1.4.1 The study of Cys703 in SceERG7

In previous study,⁴⁰ the cysteine-modifying agent, 5,5-dithio-bis-(2-nitrobenzoic) acid (DTNB), was used to assess the possible role of cysteine residues in the enzymatic function of bovine liver OSC activity. The results show that there are two cysteine residues that play important roles in catalytic function or conformation in active site of ERG7.

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Furthermore, in the sequence alignment of SceERG7, Cys703 is a highly-conserved residue in the oxidosqualene cyclases family. By homology modeling, it is a secondary sphere residue in the active-site, where the Phe699 locates between Cys703 and the substrate. The minimum distance from Cys703 to the lanosterol molecule within the SceERG7 modeling structure is 7.01 Å , while the distance between Cys703 and Phe699 is only 3.28 Å . Therefore, the mutation on Cys703 might change the situation of Phe699, and influence the mechanism of cyclization and rearrangement (Fig. 1.19). Some further studies showed the importance of second-sphere residues. The best example is the His477 within

AthCAS1, which is hydrogen bonded to Tyr410, thus it is essential for cycloartenol

Figure 1.19 The position of Cys703 in the active-site within SceERG7.

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According to the griffin aromatic hypothesis, the role of aromatic residues is considered to stabilize the highly-energy cation intermediate. The aromatic residues, which are within the putative active site, include Tyr99, Trp232, His234, Trp390, Trp443, Phe445, Tyr510, Phe699, and Tyr707. Among them, Phe699 affects the C-17 cation directly. The mutation on the Phe699 residue could produce many truncated compounds that reflect the unstable actuality of the C-17 cation. Therefore, the mutation on the Cys703 residue might indirectly influence the Phe699 residue to cause similar results.

On the other hand, the argument regarding the B-chair/boat ring and the 17α/17β mechanistic transitions has occurred lately in evolutionary history.⁴¹ The multiple products of oxidosqualene cyclases including the B-chair ring with the 17α/17β skeleton and B-boat ring with the 17β structure were isolated. Fascinatingly, no B-boat compounds with the 17α skeleton have found been in nature.⁴² So the method could be a means to understand the stereochemistry of enzymatic control.

Through the mutation on the residue Cys703 within SceERG7, the steric effect close to the C-17 cation might change. By analyzing its coresponding products, the relationship of structure-function might be understood in better detail.

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1.4.2 The study of CAS1 in A. thaliana and PSY in P. sativum

To understand the entire stereochemical mechanism of oxidosqualene cyclases, not only the information acquired from the mutation on Cys703 within SceERG7, but also the information by comparing the similarities and dissimilarities between cycloartenol synthase (CAS) and β-amyrin synthase (βAS) are needed. Either the CAS or βAS are found in higher plants. Some species, such as Arabidopsis thaliana, contain both of CAS and βAS, with distinct stero-chemical control. Many differences exist between CAS and βAS, even in the same species. CAS triggers the substrate into the chair-boat-chair conformation, forming the protosteryl cation intermediate and producing the final tetracyclic compound. βAS catalyzes the OS to go though the chair-chair-chair dammarenyl cation to form the pentacyclic product. In addition, there are some mutational studies between lupeol synthase and β-amyrin synthase as mentioned earlier, to illustrate their stero-chemical selectivity.

On the other hand, detailed information about amino acids in the active-site is critical to understand the whole mechanism of one enzyme. Just like the lanosterol cyclase from S. cerevisiae, the complete alanine-scanning of the active-site had been done by our laboratory. Our data indicated that the residue plays an important role in catalytic reaction of the enzyme, while an amino acid mutated to alanine produced novel products or lost the enzyme activity dramatically.

According to the view as mentioned above, to construct a complete site-directed alanine-scanning experiment is necessary. So based on the previous study¹⁹, many important residues were chosen in the SHC active-site. Fifteen amino acids from the SHC active-site had been chosen to find their corresponding residues in AthCAS1 and PsaPSY, then the change of the product profile was analyzed in each mutant (Fig. 1.20). The residues in CAS1 (or PSY) are V368(V371), L372(L374), W416(W418), F472(F474), C484(C486), E548(E550), F550(F552),

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I553(I555), W610(W612), Y616(Y618), F726(F728), C730(C732), I732(L734), Y734(Y736) and Y737(Y739). By mutating the residues, more information could be obtained. Based on the given information, it is possible to preliminarily predict the stereochemical mechanism of enzyme-mediated cyclization, to propose a foundation for the further studies.

Figure 1.20 The alanine-scanning mutation position in the

active-site of β-amyrin synthase within Pisum sativum.

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