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導向演化的研究
及其光親和標記的設計與合成
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計畫類別：個別型計畫
計畫編號：NSC89－2113－M－009－007－
執行期間：88 年 08 月 01 日至 89 年 07 月 31 日
計畫主持人：吳東昆
執行單位：國立交通大學生物科技研究所
中 華 民 國 89 年 10 月 27 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 89-2113-M-009-007 執行期限：88 年 8 月 1 日至 89 年 7 月 31 日 主持人：吳東昆 e-mail: [email protected] 執行機構及單位名稱：國立交通大學生物科技學系 參與計畫人員：邱逢弦，羅安堡，王幸如，謝雅如，黃喬盈 一、中文摘要 本計畫對於利用導向演化的策略以研 究氧化鯊烯環化酵素之環化反應機構已獲 得初步結果。經由利用組合基因學針對離 氨基酸-448 及白胺酸-443 將其轉變為其他 不同胺基酸的結果顯示白胺酸-443 的胺基 酸置換對於酵母菌之存活與否具有決定性 的影響。經由管柱與薄層色層分析法分離 產物的結果發現上述置換無法催化反應的 進行而停留在反應物。另一方面，經由分 子模擬的結果顯示在三度空間中白胺酸-443 與天門冬酸-456 可能分別位於受質環 氧基之兩側而在反應過程中負責穩定碳-2 上所形成的第一個帶正電的高能中間過渡 產物以幫助 A 環的合成。由以上的研究結 果，預期對增進氧化鯊烯環化酵素之環化 反應機構，以及其酵素之特定氨基酸所扮 演的結構-功能關係的了解，將有極大之助 益。 關鍵詞：氧化鯊烯，羊毛硬脂醇，定點突 變，組合基因學，導向演化，胺基丙酸掃 描法，光親和標記 AbstractThe short-term objectives towards understanding the active site architecture and cyclization mechanism of oxidosqualene
cyclase from Saccharomyces cerevisiae have
been accomplished. Two saturated mutant libraries corresponding to Lys-448 and Trp-443, respectively, were constructed and assayed for their ability to complement the
genetic disrupted ERG7 gene of S. cerevisiae
strain, CBY57, by plasmid shuffle strategy. Among the 9 non-tryptophan mutants, 7 of
them failed to complement the ERG7 disruption. Analysis of the cyclase product revealed that the cyclization reaction stopped at oxidosqualene, indicating that Trp-443 replacement might be crucial for cyclase activity. Homology modeling of the cyclase also revealed that the Trp-443, located in opposite position with Asp-456 around the epoxide of the substrate in 3-D structure, might be involved in stabilizing the high-energy C-2 cation intermediate during the
A-ring formation. In conclusion, in
combination of saturated mutations and analysis of their cyclization products as well as the molecular modeling results, would facilitate better understanding regarding the cyclization/rearrangement mechanism and structure-function relationships of amino acid residues of oxidosqualene cyclases.
Keywords: 2,3-oxidosqualene cyclase,
site-directed mutagenesis, site-directed evolution, photoaffinity label, combinatorial genetics, cassette mutagenesis, lanosterol,
cycloartenol
二、緣由與目的
The conversion of (3
S)-2,3-oxidosqualene (OS) into sterols and
tritepenes represents one of the most
remarkable and fascinating
biotransformations found in nature. The reaction is catalyzed by oxidosqualene cyclases (OSCs) which transform OS to lanosterol in mammals and fungi versus cycloartenol in algae and photosynthetic
plants. 1-4 The formation of lanosterol and cycloartenol is initiated in the pre-chair-boat-chair-boat conformation and starts from an acid-catalyzed oxirane ring opening then
followed by a series of discrete
conformationally rigid carbocationic
transformations until the metastable tricyclic
ring formation.5,6 The five membered
cyclopentenyl carbinyl cation then undergoes a ring expansion to a six membered C-ring, leading to the formation of protosterol cation.
Additional rearrangements involving
transient methyl and hydride migrations, culminated by quenching of the positive charge by deprotonation at C-9 to afford lanosterol versus by ring closure between
C-9β and C-19 to form cyclopropyl ring and
yield cycloartenol (Figur e 1).
H O H H H O H H H O H H H H H O H H H O Initiation/Cyclization H + +
Rearr an gement/Ter mination
+
Pr otost er ol Cation
:B
L an osterol Oxid osqu alene
6
A-ring B-ring C-ring
D-ring
The objectives of this research program
aimed at understanding the active site
architecture and cyclization mechanism of
oxidosqualene cyclase from Saccharomyces
cerevisiae. We have constructed two
saturated mutant libraries of Lys-448 and Trp-443, to study the relationships between
active site architecture and
cyclization/rearrangement reaction
mechanism. The effect of mutations on oxidosqualene-lanosterol cyclase activity was examined by plasmid shuffle technique
through assayed for their ability to
complement the genetic disrupted ERG7
gene of S. cerevisiae strain, CBY57. On the
other hand, we tried to design, synthesize
several substrate mimics and high-energy intermediates for site-specific labeling.
三、結果與討論
Gener ation of Satur ated Mutants in the Wild Type of S. cerevisiae ERG7 Gene.
Two saturated mutant libraries of
Lys-448 and Trp-443, respectively, were
constructed through replacement of the wild type cyclase gene with the corresponding random primers. 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. Nine
non-tryptophan and non-lysine mutants,
respectively, were identified through
sequencing the 443-448 region of the cyclase gene.
Char acter ization of Satur ated 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
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 saturated mutants,
selected for growth in the media containing adenine, lysine, histidine and uracil, then re-selected for growth in the presence of
5’-FOA. CBY57[pZS11] transformants
carrying Trp-443-Leu and Trp-443-His
mutations respectively, produced colonies upon counterselection for pZS11 with 5’-FOA. These colonies grew at a rate similar
to that observed with
CBY57[pTKOSCRS314WT]. These results clearly indicate that 443-Leu and
Trp-443-His mutants of the S. cerevisiae
oxidosqualene-lanosterol cyclase are active, and that they have activity qualitatively similar to that of the wild type enzyme. On the other hand, cyclase mutants bearing Ala, Ser, Pro, 443-Tyr, 443-Asn, 443-Thr, and
Trp-443-Arg mutations were unable to
complement the ERG7 disruption. The result indicates that above mutations either
abolish oxidosqualene-lanosterol cyclase
activity completely or reduce it below some threshold level needed to support growth of
the ERG7 knockout. In order to identify the possible intermediate generated by Trp-443 mutations, the yeast mutant strains were grown in large quantity and the ergosterol biosynthetic pathway fractions were extracted and assayed by thin-layer chromatography. Analysis of the nonsoponifiable lipid by TLC revealed that the cyclization reaction halted at oxidosqualene, indicating that Trp-443 replacement might be crucial for cyclase activity especially for the closure of the A-ring or oxirane cleavage.
Based on the assumption that the related sequences between SHC and OSC should
possess comparable 3-D structure, a
homology modeling approach was applied to determine the possible spatial location of Trp-443, in order to gain a more detailed
understanding of the structure-function
relationships. Homology modeling of the cyclase revealed that the Trp-443, located in opposite position with Asp-456 around the epoxide of the substrate in 3-D structure, might be involved in stabilizing the high-energy C-2 cation intermediate during the A-ring formation.
In summary, we have applied saturated mutagenesis and plasmid shuffle strategies and used them to determine the active site
architecture of oxidosqualene-lanosterol
cyclase. Among the 9 non-tryptophan mutants constructed, 7 of them failed to complement the ERG7 disruption. Analysis of the cyclase product revealed that the cyclization reaction stopped at oxidosqualene, indicating that Trp-443 replacement might be crucial for cyclase activity.
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. New data and insights have been obtained to facilitate the
understanding of the oxidosqualene
cyclization reaction, specially in the initiation step. Nine non-tryptophan and non-lysine mutants, respectively, were constructed and tested for their effect on oxidosqualene-lanosterol cyclase activity. The plasmid shuffle and homology modeling results of saturated mutants indicated that Lys-448 residue might be involved in the initiation of oxirane cleavage or A ring cyclization. Furthermore, Trp-443 might be involved in stabilizing the high-energy C-2 cation intermediate during the A-ring formation through spatial interaction with the Asp-456 residue located on the opposite side of the substrate.
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 A-ring formation. The scientific value of this research result provides important viewpoint relevant to understand the active site architecture and the
cyclization/rearrangement mechanism of
oxidosqualene cyclase.
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