cerevisiae ERG7 might also participate in the interaction with the resultant cyclohexyl cation due to its proper position just below the C/D ring plane (Figure 5.6, and Figure
5.2.4 Correlation of the OSC model to the experimental mutagenesis studies
Several functional residues, including Tyr-99, Trp-232, His-234, Trp-390, Trp-443, Phe-445, Lys-448, Tyr-510, Phe-699 and Tyr-707 in S. cerevisiae oxidosqualene-lanosterol cyclase (S. cerevisiae ERG7) have been respectively elucidated as the catalytically important residues for the cyclase-mediated reaction using site-directed/saturated mutagenesis coupled with bioorganic characterization in our laboratory.106, 115, 117-119, 125, 142 Diverse products including monocyclic, bicyclic, tricyclic, tetracyclic, truncated rearranged tetracyclic, altered deprotonation tetracyclic, as well as the normal biosynthetic tetracyclic product, lanosterol, were isolated from respective single amino acid substituted S. cerevisiae ERG7 mutants. These isolated premature products not only imply the plasticity of the cyclases and the conceivable evolution from the common ancestral cyclase, but also provided the direct evidence
Tyr-510
His-234 Phe-445
Lanosterol Phe-699
Tyr-99 Tyr-707
Val-4
54
Asp-456
for the existence of the long-sought mechanistic intermediate states during the biosynthesis of lanosterol.
Due to the absence of S. cerevisiae oxidosqualene-lanosterol cyclase (S.
cerevisiae ERG7) crystal structure, the elucidation of the relationship between
mutated enzyme and its product diversity is hampered. Fortunately, bioinformatic tools and quantum mechanics simulation might provide some opportunities for rationally examine the role of these functional residues in the complex cyclization/rearrangement reaction. For example, the previously created plausible S.cerevisiae ERG7 homology model based on the crystal structure of bacterial
squalene-hopene cyclase (A. acidocaldarius SHC) or human oxidosqualene-lanosterol cyclase (human OSC) could provide a suitable template for generating the individual homology modeling structures of S. cerevisiae ERG7 mutations. In addition, these individual cationic reactive intermediates or the respective annulated products including monocyclic, bicyclic, tricyclic, tetracyclic, truncated rearranged, and altered deprotonation tetracyclic structures could be simulated and docked into the active site cavity of the wild-type S. cerevisiae ERG7 and various mutated S. cerevisiae ERG7 homology models. Moreover, in consideration of the spatial orientation or electrostatic interaction between the enzymatic active site residues and the various annulated cationic intermediates, the generated homology modeled enzyme/ligand complexed structures were further applied for the energetic minimization module by Sybyl 7.0 software. The chemical structure of these cationic reactive intermediates and various annulated products is shown in Figure 5.10. Moreover, the respective premature products isolated from different mutations are listed in Figure 5.11. The examination or the local view of individual S. cerevisiae ERG7 mutation’s homology modeling structure complexed with various cationic reactive intermediates or annulated products will be discussed in the following text.Figure 5.10 The chemical structure of the possible cationic reactive intermediates and
O
ERG7ERG7Val454XThr384X ERG7Thr384X
Figure 5.11 The isolated products from various S. cerevisiae ERG7
Xmutation studies.
115-120, 125A detailed investigation of the homology modeling structures suggested that a π-electron rich pocket, which was created by previously illustrated catalytically important residues, including Tyr-99, Trp-232, His-234, Trp-390, Trp-443, Phe-445, Tyr-510, Phe-699, and Tyr-707 coupled with other aromatic amino acid residues, including Trp-587, Tyr-239, Trp-194, Phe-528, and Phe-104, existed in the S.
cerevisiae ERG7 active site. Among these functional residues, the hydrogen-bonding
network between His-234 and Tyr-510 was located on the top side of the molecular plane of lanosteryl cation, from which the Tyr-510 was spatially near the C8/C9 position, whereas His-234 was proximal to the C/D ring junction of lanosterol. In addition, the Trp-232, the neighbor residue of His-234, was thought to affect the orientation of His-234 as well as the His-234:Tyr-510 hydrogen-bond dyad.As illustrated in the previous chapter and in other published data, the single amino acid substitution of S. cerevisiae ERG7Y510X generated various products from monocyclic achilleol A or camelliol C, and altered deprotonation tetracyclic lanosterol and parkeol.125, 143 Together with the monocyclic and deprotonated structures from different S. cerevisiae ERG7Y510X mutants, the possible dual functions of this residue involved in cyclization and deprotonation steps of the oxidosqualene cyclization/rearrangement cascade has been proposed.106, 142 Moreover, from the observation of the S. cerevisiae ERG7 homology model, the hypothesized model of the stabilization of Tyr-510 residue for the monocyclic cyclization or deprotonation reaction was also discussed in the previous chapter.
The isolation of tricyclic (13αH)-isomalabarica-14(26),17,21-trien-3β-ol, tetracyclic protosta-20,24-dien-3β-ol, and protosta-12,24-dien-3β-ol from various S.
cerevisiae ERG7
H234X mutants indicated the catalytic importance of His-234 for the complex cyclization/rearrangement cascade, especially near the C/D ring junction.115,mechanistic evidence for the formation of the chair-boat 6.6.5-fused tricyclic Markovnikov cationic intermediate and chair-boat-chair 6.6.6.5-fused tetracyclic protosoteyl cation, that were assigned provisionally to the ERG7-catalyzed biosynthetic pathway. Moreover, the protosta-12,24-dien-3β-ol is the first isolated and characterized truncated rearrangement product, suggesting the important role of His-234 in stabilizing the cationic intermediate generated in the rearrangement process.115 From the observation of homology models, the position of the Nε2 of the His-234 imidazole group was found at a distance of about 4.2 Å and 4.0 Å to the C-13 and C-20 of protosteryl cations, respectively (Figure 5.12). Accordingly, the π-electron rich characteristic of His-234 was suitable and optimal for stabilizing the electron deficient cationic intermediates, especially for the chair-boat 6-6-5 tricyclic Markovnikov C-13 cation as well as the protosteryl C-20 cation. The substitution of histidine residue for other amino acids would affect the steric or electrostatic interaction between the respective cationic intermediates and enzymatic active site, and thus resulted in the generation of different ratio of achilleol A, parkeol, (13αH)-isomalabarica-14(26),17,21-trien-3β-ol, protosta-12,24-dien-3β-ol and protosta-20,24-dien-3β-ol.115, 117
(A) (B)
Figure 5.12 (A) Stereo representations of the wild-type S. cerevisiae ERG7 homology modeling structure. The putative active-site residues (stick representation) in the active-site cavity are included. Lanosterol is shown in green; while the black dotted lines show the distance between C-13 and C-20 atom of lanosterol with the Nε2 atom of His-234, respectively. (B) Partially superimposed homology modeling structures of wild-type ERG7, ERG7W232C and ERG7W232A proteins. The original His-234:Tyr-510 catalytic base dyad is shown with stick representation in gray, whereas the yellow color represents the Trp232Cys mutation and blue color represents the Ala substitution, respectively. Moreover, the protosteryl C-13 cation is also included.
On the other hand, the isolation of the same tricyclic (13αH)-isomalabarica- 14(26),17,21-trien-3β-ol from most of S. cerevisiae ERG7Y510X mutants also implied the altered orientation of His-234:Tyr-510 dyad or spatially changed enzymatic cavity. The impaired catalytic hydrogen-bonding network was further supported from the various homology models of S. cerevisiae ERG7H234X/Y510X double mutations. First, the hydrogen-bonding interaction between Tyr-510 and His-234 was exchanged for the electrostatic repulsion in the S. cerevisiae ERG7H234Y mutant, from which the orientation of Tyr-510 was away from the original position and pushed the Tyr-234 residue toward the bottom side of the enzymatic cavity, resulted in the formation of
Tyr-510
altered products. In contrast, the S. cerevisiae ERG7H234Y/Y510A mutant released the electrostatic repulsion and restored the partial active site cavity, which resulted in the production of lanosterol as its sole product (Figure 5.13). Consistently, from the recent experimental results of the S. cerevisiae ERG7H234W/Y510V mutant that produced the polycyclic products like lanosterol and (13αH)-isomalabarica-14(26), 17,21-trien-3β-ol; whereas the ERG7H234W/Y510W mutant that produced only monocyclic achilleol A, further indicated the spatial importance of Tyr-510 residue in the cyclization reaction. The polycyclic products isolated from the smaller substitution of Tyr-510 in either the S. cerevisiae ERG7H234W/Y510V mutant or previous S. cerevisiae ERG7H234Y/Y510A indicated the catalytical importance of His-234 for stabilizing the C-13 and C-20 protosteryl cations respectively. The partial disturbance of the active site residues especially near the C-13 or C-20 of protersteryl cations was observed when the His-234 was changed into other amino acid. The destabilization of the corresponding cationic intermediates resulted in generation of the alternative deprotonated products. On the contrast, the isolation of monocyclic achilleol A from the S. cerevisiae ERG7H234W/Y510W mutant or S. cerevisiae ERG7Y510W mutant suggested that the bulky substitution of Tyr-510 dramatically pushed the His-234 away from the original position, blocked the polycyclization progress after the A-ring formation, and resulted in the truncated monocyclic product production. The steric effect of the bulky tryptophan residue, the affected cavity conformation, as well as the coordinative interaction between Tyr-510 and His-234 can be apparently observed from the homology modeling structure of S. cerevisiae ERG7 (Figure 5.13).
(A) S. c. ERG7H234Y mutant (B) S. c. ERG7H234Y/Y510A double mutant
Figure 5.13 The Stereo representations of various S. cerevisiae ERG7 mutations modeling structures. (A) ERG7H234Y mutant (B) ERG7H234Y/Y510A double mutant (C) The partially superimposed ERG7 and ERG7H234W/Y510W mutant models (D) The partially superimposed ERG7 and ERG7H234W/Y510V mutant models.
Ala-510
(C) The wild-type ERG7 (gray) and ERG7H234W/Y510W mutant (yellow)
(D) The wild-type ERG7 (gray) and ERG7H234W/Y510V mutant (blue)
Tyr-510
On the other hand, the catalytic importance of the Trp-232 might be implied via the indirect interactions from the spatially affected active site residues. From the homology models illustration, the orientation of the hydrogen-bonding network of the His-234:Tyr-510 catalytic base dyad was slightly moved after substituting the bulky Trp-232 residues to other amino acids. Obviously, the distance from His-234 to the protosteryl C-13 cation was changed and slightly close toward the C-11 or C-12 position of lanosteryl cation. Thus, the alternatively deprotonated products, either parkeol or protosta-12,24-dien-3β-ol, were respectively generated in the replacement of the bulky Trp-232 residue to the smaller residues (Figure 5.12).118
The tricyclic (13αH)-isomalabarica-14(26),17,21-trien-3β-ol, and three altered deprotonation products: lanosterol, parkeol, and 9β-lanosta-7,24-dien-3β-ol were also observed from the products profile in the S. cerevisiae ERG7F445X mutants.119 The careful examination of the mutants' homology models suggested that the substitution of the S. cerevisiae ERG7F445X significantly influenced the cavity size and steric orientation of the π-electron rich active site pocket. The homology models of S.