CYP72A67 Catalyzes a Key Oxidative Step in Medicago truncatula Hemolytic Saponin Biosynthesis
CYP72A67 Catalyzes a Key Oxidative Step in Medicago truncatula Hemolytic Saponin Biosynthesis
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DOI：
10.1016/j.molp.2015.06.003
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发表时间：
2015-10-05
期刊：
影响因子：
27.5
通讯作者：
Calderini, Ornella
中科院分区：
文献类型：
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作者：
Biazzi, Elisa;Carelli, Maria;Calderini, Ornella
在苜蓿属中，三萜皂苷是通过类异戊二烯途径在植物的地上和地下部分组成性合成的生物活性次级代谢产物。皂苷作为药物、农用化学品以及在食品和化妆品工业中的开发引起了人们对鉴定参与其合成的酶的兴趣。我们已经确定了一个细胞色素P450（CYP 72 A67）参与溶血皂苷元生物合成的反向遗传TILLING方法在蒺藜苜蓿乙基甲磺酸盐（EMS）诱变收集。遗传和生物化学分析，突变互补，并在微粒体酵母系统中的基因的表达表明，CYP 72 A67是负责羟基化的C-2位下游的油酸合成。在相同的体外酵母系统中，研究了CYP 72 A67对多个碳位置上具有不同取代的底物的亲和力，以及与负责产生Medicagenic酸的另外两个CYP 450（CYP 72 A68）的关系，Medicagenic酸是M中的主要皂苷元。蒺藜叶和根。全同胞突变体和野生型植物进行了比较，他们的皂苷元的配置文件，参与皂苷元合成的基因的表达模式，接种苜蓿中华根瘤菌的反应。所得结果使我们能够修改M. truncatula和有助于突出皂苷元合成的组织特异性（叶/根）。
In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 (CYP72A67) involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethylmethanesulfonate (EMS) mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstream of oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s (CYP72A68) responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities (leaves/roots) of sapogenin synthesis.