Mutational studies on triterpene syntheses:: Engineering lupeol synthase into β-amyrin synthase
Mutational studies on triterpene syntheses:: Engineering lupeol synthase into β-amyrin synthase
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DOI：
10.1021/ja0010709
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发表时间：
2000-07-26
影响因子：
15
通讯作者：
Ebizuka, Y
中科院分区：
文献类型：
--
作者：
Kushiro, T;Shibuya, M;Ebizuka, Y
对β-香树脂醇（PNY）和羽扇豆醇（OEW）这两种三萜糖苷酶进行了定点诱变，以鉴定负责其产物特异性的氨基酸残基。除了已知氧化角鲨烯环化酶之间的序列比较之外，我们先前的嵌合研究表明，β-香树脂素合酶PNY的（MWCYCR 263）-M-258序列（羽扇豆醇合酶OEW的（MLCYCR 260）-M-255序列）将参与产物分化。为了检验该假设，将Trp 259（PNY的MWCYCR）突变为Leu（PNY W259 L突变体）。在酵母中的功能性表达和产物分析显示，该突变体以2：1的比例与β-香树脂素一起产生羽扇豆醇作为主要产物。还生产了一些其他次要产品，包括丁子醇。另一方面，Leu 256（OEW的MLCYCR）突变为Trp（OEW L256 W突变体）。该突变体仅产生具有少量羽扇豆醇的β-香树脂醇，表明单个突变已将羽扇豆醇合酶工程改造成β-香树脂醇合酶。因此，β-香树脂素合成酶的Trp 259被鉴定为控制β-香树脂素形成的残基，推测其通过稳定油酰基阳离子来实现，而Leu残基缺乏这种作用可能会在羽扇烯基阳离子阶段终止反应。在进一步的突变研究中，将在所有产生五环三萜的OSC中保守的Tyr残基（PNY中的MWCYCR和OEW中的MLCYCR）突变成在所有产生四环碳骨架的OSC中发现的His，以研究PNY的该Tyr 261的作用。PNY Y261 H突变体产生达玛拉-18，21-二烯-3 β-醇（作为在Δ 18处的E/Z异构体的3：5混合物）以及少量的达玛拉-18（28），21-二烯-3 β-醇，表明β-香树脂素合酶的Tyr 261在产生五环三萜中起重要作用，推测是通过稳定达玛烯基阳离子之后产生的阳离子中间体之一。
Site-directed mutagenesis was carried out on two triterpene synthases, beta-amyrin (PNY) and lupeol (OEW) synthases, to identify the amino acid residues responsible fbr their product specificity. In addition to sequence comparison among known oxidosqualene cyclases, our previous chimeric studies suggested that (MWCYCR263)-M-258 sequence of beta-amyrin synthase PNY ((MLCYCR260)-M-255 sequence of lupeol synthase OEW) would participate in product differentiation. To test this hypothesis, Trp259 (MWCYCR of PNY) was mutated to Leu (PNY W259L mutant). Functional expression in yeast and product analysis revealed that this mutant produced lupeol as a major product together with beta-amyrin in 2:1 ratio. Some other minor products including butyrospermol were also produced. On the other hand, Leu256 (MLCYCR of OEW) was mutated to Trp (OEW L256W mutant). This mutant produced exclusively beta-amyrin with only minor amount of lupeol, demonstrating that a single mutation had engineered lupeol synthase into beta-amyrin synthase. Therefore, Trp259 of beta-amyrin synthase was identified to be the residue controlling beta-amyrin formation presumably through stabilization of oleanyl cation, while lack of this effect by Leu residue may terminate the reaction at lupenyl cation stage. In further mutation studies, Tyr residue (MWCYCR in PNY and MLCYCR in OEW) conserved in all of the OSCs producing pentacyclic triterpenes was mutated into His which is found in all of those producing tetracyclic carbon skeletons to investigate the role of this Tyr261 of PNY. PNY Y261H mutant produced dammara-18,21-dien-3 beta-ol (as a 3:5 mixture of E/Z isomer at Delta 18) together with a minor amount of dammara-18(28),21-dien-3 beta-ol, demonstrating that Tyr261 of beta-amyrin synthase plays an important role in producing pentacyclic triterpenes presumably by stabilizing one of the cation intermediates generated after dammarenyl cation.