Functional Identification of Triterpene Methyltransferases from Botryococcus braunii Race B
Functional Identification of Triterpene Methyltransferases from Botryococcus braunii Race B
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DOI：
10.1074/jbc.m111.316059
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发表时间：
2012-03-09
影响因子：
4.8
通讯作者：
Chappell, Joe
中科院分区：
文献类型：
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作者：
Niehaus, Tom D.;Kinison, Scott;Chappell, Joe
布朗葡萄球菌小种B是一种集落形成的绿色藻类，其积累的三萜油超过其干重的30%。三萜油的组成主要由葡萄球菌烯和角鲨烯的二甲基化至四甲基化形式。虽然最近描述了葡萄球菌和角鲨烯生物合成的不寻常机制，但负责用甲基取代基装饰这些三萜支架的酶是未知的。B的转录组。通过计算筛选braunii，假设三萜甲基转移酶（TMT）可能类似于描述的用于甾醇侧链甲基化的S-腺苷甲硫氨酸依赖性酶。六甾醇甲基转移酶样基因的分离和功能的特点。当这些基因中的三个与互补的角鲨烯合酶或葡萄球菌合酶表达盒在酵母中共表达时，导致两种三萜支架的单和二甲基化形式的积累。令人惊讶的是，TMT-1和TMT-2表现出对角鲨烯作为甲基受体底物的偏好，而TMT-3表现出对葡萄球菌作为其甲基受体底物的显著偏好。这些在体内的偏好，证实了在体外试验中使用微粒体制剂从酵母过表达各自的基因，编码膜相关酶。通过NMR对体内酵母产生的单和二甲基化产物进行结构检查，确定了末端碳C-3和C-22/C-20分别作为甲基添加到角鲨烯和葡萄球菌的原子受体位点。这些网站是相同的那些以前报道的三萜提取的藻类。表现出不同的底物选择性和连续的催化活性的密切相关的三萜甲基转移酶的可用性提供了重要的工具，调查的分子机制负责这些独特的酶所表现出的特异性。
Botryococcus braunii race B is a colony-forming, green algae that accumulates triterpene oils in excess of 30% of its dry weight. The composition of the triterpene oils is dominated by dimethylated to tetramethylated forms of botryococcene and squalene. Although unusual mechanisms for the biosynthesis of botryococcene and squalene were recently described, the enzyme(s) responsible for decorating these triterpene scaffolds with methyl substituents were unknown. A transcriptome of B. braunii was screened computationally assuming that the triterpene methyltransferases (TMTs) might resemble the S-adenosyl methionine-dependent enzymes described for methylating the side chain of sterols. Six sterol methyltransferase-like genes were isolated and functionally characterized. Three of these genes when co-expressed in yeast with complementary squalene synthase or botryococcene synthase expression cassettes resulted in the accumulation of mono-and dimethylated forms of both triterpene scaffolds. Surprisingly, TMT-1 and TMT-2 exhibited preference for squalene as the methyl acceptor substrate, whereas TMT-3 showed a striking preference for botryococcene as its methyl acceptor substrate. These in vivo preferences were confirmed with in vitro assays utilizing microsomal preparations from yeast overexpressing the respective genes, which encode for membrane-associated enzymes. Structural examination of the in vivo yeast generated mono-and dimethylated products by NMR identified terminal carbons, C-3 and C-22/C-20, as the atomic acceptor sites for the methyl additions to squalene and botryococcene, respectively. These sites are identical to those previously reported for the triterpenes extracted from the algae. The availability of closely related triterpene methyltransferases exhibiting distinct substrate selectivity and successive catalytic activities provides important tools for investigating the molecular mechanisms responsible for the specificities exhibited by these unique enzymes.