行政院國家科學委員會專題研究計畫 成果報告
氧化鯊烯環化酵素之產物多樣性與開發寡核酸先導藥物之
研究
計畫類別： 個別型計畫 計畫編號： NSC91-2113-M-009-023- 執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日 執行單位： 國立交通大學生物科技研究所 計畫主持人： 吳東昆 計畫參與人員： 張程翔，柯震宇，張琬琳，陳京瑤，劉媛婷，王裕國 報告類型： 精簡報告 處理方式： 本計畫可公開查詢中 華 民 國 92 年 10 月 29 日
行政院國家科學委員會補助專題研究計畫成果報告
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氧化鯊烯環化酵素之產物多樣性作用機制探討與開發寡核酸先導藥物之研
究
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計畫類別：■個別型計畫 □整合型計畫
計畫編號：NSC 91－2113－M－009－023－
執行期間：91 年 08 月 01 日至 92 年 07 月 31 日
計畫主持人：吳東昆
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
□出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
執行單位：國立交通大學生物科技學系
中 華 民 國 92 年 10 月 29 日
行政院國家科學委員會專題研究計畫成果報告
計畫編號：NSC 91－2113－M－009－023－
氧化鯊烯環化酵素之產物多樣性與開發寡核酸先導藥物之研究
執行期限：91 年 08 月 01 日至 92 年 07 月 31 日
主持人：吳東昆 國立交通大學生物科技學系
計畫參與人員：張程翔，柯震宇，張琬琳，陳京瑤，劉媛婷，王裕國 一、中文摘要 本計畫對於研究氧化鯊烯環化酵素之產物多 樣性與其環化反應機制間之關係已獲得初步結 果。經由針對由阿拉伯芥之氧化鯊烯環化酵素 (CAS)進行雙點突變以觀察其功能之變化與演化 之關係，並進一部探討其環化/重組機制、以及產 物之專一性與多樣性。結果顯示在 29 個 針對酵 素活性區域表面之胺基丙酸突變株中有 16 個突 變株經置換後無法獲得羊毛硬脂醇之產物以互補 酵母菌之生存。這 29 個突變株中Y118、L124、 G127、L179、T215、G366、P367、V368、L372、 C484、E548、Y616 與 C730，對於 CAS 環化重 組的機制沒有顯著的影響；F123、W217、W221、 M254、H257、V261、Y262、W416、F472、F550、 I553、W610、F726、I732、Y734 與 Y737 在 CAS 催化的過程中，可能協助受質環化與重組，擔任 決定酵素活性有無的關鍵角色。 在分析CAS 含雙點突變之產物的過程中，利 用打破具雙點突變之酵母菌，以萃取的方式從酵母 菌細胞中擷取出非可皂化的脂質。利用薄層色層分 析片 ( TLC ) 了解產物的分佈、以矽膠的管柱 ( Silica column ) 將產物纯化分離、氣相色層分析 ( GC-MS ) 推其分子量，再利用核磁共振光譜學 ( NMR ) 分析產物的結構。由產物分析結果發現， 包 含 活 性 區 域 靠 近 受 質 接 受 區 下 方 之 M254、 H257、V261 的雙點突變效應下，有相同的新產物 生 成 。 新 產 物 分 別 是 24-Methlene 24,25 -Dihydrolanosterol 、 4,4-Dimethyl fecosterol 與 4-Methyl Fecosterol，分子量分別為 440、 426 與 412。我們推測 CASY410C M254A、CASY410C H257A 與CASY410C V261A 突變效應，失去環化的活性而轉變成
固 醇 類 甲 基 轉 移 酶 的 功 能 性 ( Sterol Methyl Transferase, SMT )，使得羊毛硬脂醇改變代謝途徑
產生 24-Methlene 24, 25-Dihydrolanosterol，隨即經 細 胞 中 C-14α 的 去 甲 基 酶 作 用 後 ， 進 而 形 成 4,4-Dimethyl Fecosterol 、 4-Methyl Fecosterol 與 Fecosterol，進入野生型麥角脂醇生合成途徑的下 游，最後形成終產物麥角脂醇 ( Ergosterol )。
關鍵詞：分子模擬，胺基丙酸掃描，定點突變，氧 化鯊烯環化酵素，羊毛硬脂醇，重複質譜
Abstract
Oxidosqualene-lanosterol cyclase (OSC) and oxidosqualene-cycloartenol synthase (CAS) catalyze the complex
cyclization/rearrangement of (3S)2,3-oxidosqualene into lanosterol in
mammal and fungi versus cycloartenol in algae and photosynthetic plants. To study evolutionary relationships between OSC and CAS, cyclization / rearrangement mechanism, as well as product specificity and/or diversity, twenty-nine non-alanine residues located on the putative active site cavity surface of CASY410C mutant, a CAS mutant which changes its enzymatic activity from CAS to OSC, were mutated to alanine and assayed for their ability to complement the OSC deficiency and to study the effect of double mutations. All of the mutations were verified by restriction mapping and DNA sequencing, followed by genetic counter-selection. Among them, 16 out of 29 mutations failed to complement the ERG7 disruption indicating that these residues are crucial for
the catalytic function of the enzyme.
The nonsaponifiable lipids of inactive mutants were isolated and characterized by TLC, GC-MS and NMR. The CASV261AY410C mutant 、 CASM254AY410C mutant and CASH257AY410C mutant, which alone failed to complement the viability of cyclase-deficient yeast strain but accumulated 24-methylene-24,25-dihydrolanosterol
(eburicol), 4,4-dimethyl fecosterol and 4-methyl fecosterol, when both wild-type OSC and CAS double mutations were present, were isolated. The results indicated that a dramatic enzymatic activity change from oxidosqualene cyclase to sterol methyl transferase and concomitantly generated an alternative ergosterol biosynthetic pathway, were derived from the above-mentioned double mutations. In addition, the discovery here will provide important information with which to understand the active site topology for both enzymes and have significant consequences in relation to rational drug design and to the molecular engineering of the ergosterol biosynthetic pathway.
The family of triterpene cyclase enzymes catalyzes highly diverse and complex cyclization/rearrangement reactions (1-3). In fungi and mammals oxidosqualene-lanosterol cyclase (ERG7, EC
5.4.99.7) mediates the conversion of (3S)-2,3-oxidosqualene into lanosterol,
whereas this same substrate is elaborated to cycloartenol through the agency of oxidosqualene-cycloartenol synthase (CAS, EC 5.4.99.8) in algae and photosynthetic plants. In prokaryotes, squalene-hopene cyclase (SHC) catalyzes the conversion of squalene to pentacyclic triterpenoids (Fig. 1).
Keywords: oxidosqualene-lanosterol cyclase, site-directed mutagenesis, plasmid shuffling, cation-π interaction, alanine-scanning, tandem-MS-MS.
二、緣由與目的
This enzymatic reaction represents one of the most remarkable and fascinating biotransformations in nature and has inspired many scientists to study its structure-function correlations (4-12). Moreover, over 90 triterpenes with C30H50O formula have been derived from (oxido)squalene cyclization (13). In parallel, the oxidosqualene cyclases are also attractive targets in designing antifungal and anticholesteremic drugs (14-18). Elegant biogenetic and bioorganic investigations, including studies of the structures of novel products formed from designed alternate substrates, have provided a detailed model for cyclase-mediated substrate transformation (19-25). In this model, enzymes initiate cyclization cascades by protonating the terminal double bond of squalene or the oxirane moiety of oxidosqualene. The enzymes then promote cyclization cascades leading, in the case of ERG7 and CAS, to an intermediate protosterol cation which undergoes a series of hydride and methyl shifts. Lanosterol
O Lanosterol HO HO Cycloartenol H H H H H H Squalene-Hopene Cyclase Hopene Squalene Monooxygenase Oxidosqualene Squalene H
results from C-9 deprotonation of a C-8 cation while cycloartenol forms after a further hydride migration and deprotonation of the C-10 methyl group (Figure 2)
H O H H H O H H H O H H H H H O H H H O Initiation/Cyclization H + + Rearrangement/Termination + Protosterol Cation :B Lanosterol Oxidosqualene 6 A-ring B-ring C-ring D-ring Figure 2. Complex Cyclization/Rearrangement of Oxidosqualene to Lanosterol.
The speculation of the parallel active site sueface location may also be important in determining substrate recognition, cyclization/rearrangement cascade, or changing product specificity for all cyclases, the corresponding region in S.
cerevisiae was subjected to investigation.
Threfore, twenty-nine non-alanine residues located on the putative active site surface of CAS from A. thaliana were also mutated and assayed for their ability to complement the cyclase-deficient, ERG7 knockout, S.
cerevisiae strain. These results will
further extend our understanding about the mechanism of oxidosqualene cyclization/rearrangement cascade and provide basis for further elucidation of the biological origins as well as to develop novel OSC inhibitors for antifungal and hypocholesteremic purposes.
三、結果與討論
Generation of Active Site
Site-Directed Mutants in the Single Mutant of A. thaliana cas Gene.
Twenty-nine non-alanine residues were constructed through replacement of the single mutant cyclase gene with the corresponding alanine substitution. Mutants were generated by polymerase chain reaction (PCR), restriction digested with endonuclease restriction enzyme, followed by cloning into a vector to obtain a recombinant plasmid containing a single DNA fragment incorporating the desired mutation sequence and the new restriction site (Table 1). All of the mutant clones were confirmed through DNA sequencing by using ABI PRISM BigDye Terminator Cycle Sequencing Reaction kit on an Applied Biosystems 373 DNA Sequencer available in the Institute.
Characterization of Site-Directed Mutagenesis Effects on Oxidosqualene Cyclase Activity via Plasmid Shuffle.
Haploid strain CBY57[pZS11] (ERG7 △::LEU2 ade2-101 his3- △ 200 leu2- △ 1 lys2-801 trp1-△63 [pZS11]), a yeast strain bearing both a genomic ERG7-disrupted gene and a URA3 centromeric plasmid with wild type S. cerevisiae cyclase gene, allowed the use of a plasmid shuffle to analyze the effects of mutations in an ERG7 knockout background. As negative and positive controls for the plasmid shuffle, CBY57[pZS11] was transformed by electroporation with plasmids pRS314 and pTKOSCRS314WT, TRP1 centromeric plasmids bearing no insert and the wild type
S. cerevisiae oxidosqualene-lanosterol
cyclase gene, respectively. Transformants were selected on SD media containing adenine, lysine, histidine and uracil, and pRS314 and pTKOSCRS314WT were recovered from cell-free lysates grown in this
media. CBY57[pZS11], CBY57[pZS11][pRS314] and
CBY57[pZS11][pTKOSCRS314WT] were then plated on complete media containing 5-fluoroorotic acid (5’-FOA) to counterselect for pZS11. As expected, growth was only observed on plates inoculated with CBY57[pZS11][pTKOSCRS314WT].
CBY57[pZS11] was transformed by electroporation with 29 site-directed CAS mutants and selected for growth in the media containing adenine, lysine, histidine and uracil, then re-selected for growth in the presence of 5’-FOA. Following the counterselection with 5’-FOA, F123A 、 W217A、W221A、M254A、H257A、V261A、 Y262A、W416A、F472A、F550A、I553A、 W610A、F726A、I732A、Y734A and Y737A mutations failed to complement the ERG7 disruption, indicating that these residues are crucial for the catalytic function of the enzyme (Table 1). pWLC_RS313CAS***AY410C NMR GC mass New product W2 = pWLC2RSCAS F123AY410C ˇ ˇ - W7 = pWLC7RSCASW217AY410C ˇ ˇ - W8 = pWLC8RSCASW221AY410C ˇ ˇ - W9 = pWLC9RSCASM254AY410C ˇ ˇ ˇ W10 = pWLC10RSCASH257AY410C ˇ ˇ ˇ W11 = pWLC11RSCAS V261AY410C ˇ ˇ ˇ W12 = pWLC12RSCAS Y262AY410C ˇ ˇ - W17 = pWLC17RSCAS Y410CW416A ˇ ˇ - W18 = pWLC18RSCASY410C F472A ˇ ˇ -
W21 = pWLC21RSCAS Y410C F550A ˇ ˇ -
W22 = pWLC22RSCAS Y410C I553A ˇ ˇ -
W23 = pWLC23RSCAS Y410CW610A ˇ ˇ -
W25 = pWLC25RSCAS Y410C F726A ˇ ˇ -
W27 = pWLC27RSCAS Y410C I732A ˇ ˇ -
W28 = pWLC28RSCAS Y410C Y734A ˇ ˇ -
W29 = pWLC29RSCAS Y410C Y737A ˇ ˇ -
Wild type CAS ˇ ˇ Cycloartenol
Wild type OSC (CBY57) ˇ ˇ Lanosterol
Table 1. Complementation results of
site-directed mutations of double mutations of oxidosqualene-cycloartenol synthase gene in CBY58 strain.
Structure Characterization of New Products Produced by CAS Double Mutants We generated site-specific
mutants and performed plasmid shuffle selection to identify amino acid residues crucial for cyclization/rearrangement mechanism and product diversity, as previously described.2,3 Among several inactive mutants identified, the products distribution of W9, W10, W11 and W25 within yeast strain, which contains both wild type ERG7 and CAS double mutant within the strain, were analyzed. Preliminary GC revealed three new membraneous products, in addition to ergosterol. Two of these products showed identical chromatographic migration on TLC to that of lanosterol (LA), and one migrated between LA and ergosterol. Following isolation by column chromatography and characterization by GC-MS, a new major compound was identified that has identical molecular mass (m/z = 426) but has a different structure compared to LA. The 150 MHz 13C NMR spectrum of this compound revealed the presence of one di-quaternary substituted and one quaternary-quaternary substituted double bonds ( 105.93 156.67 and 127.82, 135.79 ppm). The 600 MHz 1H NMR spectrum also showed the presence of olefinic protons at 4.67 and four quaternary methyl groups ( 0.59, 0.80, 0.97, and 0.98, each s, 3H). The HMQC spectrum showed that the methyl protons at 0.59 are attached to the carbon at 11.22 ppm and that the methyl protons at 0.80 are attached to the carbon at 15.35 ppm. For the olefinic protons, the HMQC
spectrum established that the methylene protons at 4.67 are attached to the carbon at 105.93 ppm. However, detailed structure of the major compound could not be elucidated with 1H and 13C NMR, DEPT, HMBC, HMQC, and even INADEQUATE spectra. A crystal subjected to X-ray diffraction analysis revealed the three-dimensional structure to be 4,4-dimethyl fecosterol (4,4-DMF), as shown in Figure 3.
Fig. 3. Structure of 4,4-dimethyl fecosterol as determined by X-ray diffraction. Selected distances (Å): C(8)-C(9) = 1.341;
C(24)-C(28) = 1.380; C(3)-O(1) = 1.436.
Various amounts of another compound, which showed the M+ at m/z = 440, were also detected from W9, W10, W11 and W25 mutants. GC-MS analysis and comparison to published data revealed the trace
compound to be 24-methylene-24,25-dihydrolanosterol
(eburicol).5,6 Analysis of the 1H and 13C NMR signals for the minor component revealed that this compound also contains two double bonds and is one carbon less than that of 4,4-DMF, implying that this compound was possibly derived from 4,4-DMF. Detailed analysis of the 1H and 13C NMR, DEPT, HMBC, HMQC spectra as well as comparison to that of 4,4-DMF confirmed the identification of this compound as 4-methyl fecosterol (4-MF). The W25 mutant containing both Y410C and F726A produced lanosterol in conjunction with eburicol, 4,4-DMF and 4-MF as shown in Fig. 4. 1：5 m/z = 440 m/z = 426 CASM254AY410C 1：5 m/z = 440 m/z = 426 CASM254AY410C 1：7 m/z = 426 m/z = 440 CASH257AY410C 1：7 m/z = 426 m/z = 440 1：7 m/z = 426 m/z = 440 CASH257AY410C 1.35：1 m/z = 440 m/z = 426 CASV261AY410C 1.35：1 m/z = 440 m/z = 426 CASV261AY410C m/z = 426 m/z = 440 0.2 : 1：1 CASY410CF726A m/z = 426 m/z = 440 0.2 : 1：1 CASY410CF726A
Figure 4. GC spectra of W9, W10, W11, and W25.
In S. cerevisiae, the consensus opinion for LA conversion to ergosterol has been that three consecutive rounds of demethylation occurred at the C-14 and C-4 positions of LA, yielding zymosterol. This is then methynylated by ∆24(25) sterol methyl transferase (ERG6) to produce fecosterol.5,9 Analyses of sterol composition of S.
cerevisiae also revealed the presence of LA,
zymosterol, fecosterol, episterol, and ergosterol, but not the C-31 sterol derivative eburicol.5,10 The main sterols accumulated in this study are also different from that
obtained from fission yeast
Schizosaccharomyces pombe and Schizosaccharomyces octosporus, as well as
from ketoconazole-treated Trypanosoma
cruzi, all of which accumulate eburicol as a
major end product.5,11 Thus, the isolation of eburicol, 4,4-DMF, and 4-MF in this study was seemingly inconsistent with previous observation for S. cerevisiae ergosterol biosynthetic pathway. However, this can be resolved if both an additional route from LA to ergosterol exists and a new sterol methyl transferase activity occurred upon CAS double mutant transformation of the CBY57[pZS11] strain. In the presence of both ERG7 and CAS double mutants, two pathways may exist from LA to fecosterol, as shown in Fig. 5. 1) In the classical pathway, LA is demethylated at positions C-14 and C-4 to yield zymosterol and then methynylated by ERG6 to produce fecosterol. 2) In the alternative pathway, the formation of the eburicol from LA is followed by consecutive C-14 and C-4 demethylations to yield 4,4-DMF and 4-MF, followed by a third demethylation to yield fecosterol.
O HO H Lanosterol (LA) HO H HO H HO H HO H HO H HO H HO H ERG25 ERG25 Oxidosqualene (OS) 4,4-dimethyl
Zymosterol Zymosterol4-methyl Zymosterol
eburicol 4,4-dimethyl fecosterol
(4,4-DMF) 4-methyl fecosterol (4-MF) Fecosterol ERG7 /ER G7F445A ERG7 F445A ERG11 ERG24 ERG6 ERG11 ERG24 ERG25 ERG25 ERG7
Figure 5. Proposed sterol
transformations and encoding genes, from oxidosqualene to fecosterol, in the
presence of ERG7 or ERG7/ERG7F445A, in
Saccharomyces cerevisiae. ERG7, oxidosqualene-lanosterol cyclase; ERG11,
lanosterol C-14 demethylase; ERG24, sterol C-14 reductase; ERG25, sterol C-4 methyl oxidase; ERG6, sterol C-24 methyl
transferase.
Detailed comparison of putative reaction mechanism and active site topography revealed high similarities between oxidosqualene cyclase and sterol methyl transferase.3a,12,13 Both enzymes are
commonly hypothesized to form a stereochemically prefolded substrate conformation and then generate a carbocation intermediate, which undergoes 1,2-hydride migration and deprotonation. Analysis of protein sequences for both enzymes also revealed the aromatic amino acids involved in substrate binding or catalysis. The phenylalanine to alanine substitution may cause minor changes in the topography of the active site and affect the substrate (or product) affinity, the relationship between binding energy and catalysis, or the recognition of the terminal side chain. An alternative biosynthetic pathway to ergosterol from lanosterol was then generated.
In conclusion, we identified four CAS double mutants that fail to complement the viability of cyclase-deficient yeast strain but accumulates eburicol, 4,4-DMF and 4-MF when present with wild type ERG7. These CAS mutants derived from A. thaliana, which was demonstrated for the first time in
S. cerevisiae, showed that a double amino
acid residues substitution in the primary sequence of oxidosqualene cyclase can alter the ergosterol biosynthetic pathway and result in the accumulation of novel 24-methylene sterols end products. Finally, this discovery may provide important information for understanding the active site topology for both CAS and ERG6 enzymes and significantly benefit rational drug design and molecular engineering of the ergosterol biosynthetic pathway.
四、計劃成果自評
We have successfully accomplished the preliminary objective to study the active site architecture and cyclization/rearrangement
reaction mechanism of oxidosqualene-lanosterol cyclase as well as
to expand cyclase product diversity. New data and insights have been obtained to facilitate the understanding of the oxidosqualene cyclization reaction, specially in the initiation step and substrate specificity and protein evolution. Based on the active site residues site-directed mutagenesis results and novel product isolation, an evolutionary model for the oxidosqualene cyclase enzyme could be proposed. The obtained result has been advanced in elucidating the possible role of some amino acid residues present in the enzyme, specially for the residues involved in the initiation of cyclization or high energy transition state intermediate. Therefore, the scientific value of this research result provides important viewpoint relevant to understand the active site
architecture and the cyclization/rearrangement mechanism of
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