One amino acid makes the difference: the formation of ent-kaurene and 16α-hydroxy-ent-kaurane by diterpene synthases in poplar.
One amino acid makes the difference: the formation of ent-kaurene and 16α-hydroxy-ent-kaurane by diterpene synthases in poplar.
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DOI：
10.1186/s12870-015-0647-6
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发表时间：
2015-10-28
影响因子：
5.3
通讯作者：
Köllner TG
中科院分区：
文献类型：
--
作者：
Irmisch S;Müller AT;Schmidt L;Günther J;Gershenzon J;Köllner TG
与劳丹相关的二萜类化合物形成了二萜类化合物中最大的组。它们作为必需的植物生长激素在初级代谢中发挥重要作用，并且已知在次级代谢中发挥作用，例如植物抗毒素。劳丹相关二萜的生物合成由II类和I类二萜脱氢酶的作用介导。虽然萜烯脱氢酶在白杨中已经得到了很好的研究，但对这种木本多年生植物中二萜的形成知之甚少。最近测序的大果杨基因组具有两个推定的柯巴基二磷酸合成酶基因（CPS，II类）和两个推定的贝壳杉烯合成酶基因（KS，I类），这最有可能产生通过基因组重复和最近的串联基因重复，分别。我们发现，CPS-样基因PtTPS 17编码一个ent-copalyl二磷酸合酶（ent-CPS），而由推定的CPS基因PtTPS 18编码的蛋白质没有表现出酶活性。推测的贝壳杉烯降解酶PtTPS 19和PtTPS 20均以对映-柯巴基二磷酸（ent-CPP）为底物。然而，尽管它们的高序列相似性，它们产生不同的二萜产物。PtTPS 19只生成对映贝壳杉烯，而PtTPS 20主要生成二萜醇16α-羟基对映贝壳杉烷。使用基于同源性的结构建模和定点突变，我们证明了一个氨基酸残基决定了PtTPS 19和PtTPS 20的不同产物特异性。甲硫氨酸607和苏氨酸607在PtTPS 19和PtTPS 20的活性位点的相互交换，分别导致酶产物谱的完全相互转换。基因表达分析表明，二萜合酶基因在白杨根中表现出器官特异性表达，PtTPS 17和PtTPS 20转录本丰度最高。白杨二萜降解酶PtTPS 17、PtTPS 19和PtTPS 20在白杨中参与对映贝壳杉烯和16α-羟基对映贝壳杉烷的生成。虽然对映贝壳杉烯很可能是赤霉素的通用前体，但16α-羟基-对映贝壳杉烷在白杨中的功能尚不清楚。PtTPS 20和PtTPS 17在白杨根中的高表达可能表明16α-羟基贝壳杉烷在杨树根的次生代谢中起重要作用。本文的在线版本（doi：10.1186/s12870-015-0647-6）包含补充材料，可供授权用户使用。
Labdane-related diterpenoids form the largest group among the diterpenes. They fulfill important functions in primary metabolism as essential plant growth hormones and are known to function in secondary metabolism as, for example, phytoalexins. The biosynthesis of labdane-related diterpenes is mediated by the action of class II and class I diterpene synthases. Although terpene synthases have been well investigated in poplar, little is known about diterpene formation in this woody perennial plant species. The recently sequenced genome of Populus trichocarpa possesses two putative copalyl diphosphate synthase genes (CPS, class II) and two putative kaurene synthase genes (KS, class I), which most likely arose through a genome duplication and a recent tandem gene duplication, respectively. We showed that the CPS-like gene PtTPS17 encodes an ent-copalyl diphosphate synthase (ent-CPS), while the protein encoded by the putative CPS gene PtTPS18 showed no enzymatic activity. The putative kaurene synthases PtTPS19 and PtTPS20 both accepted ent-copalyl diphosphate (ent-CPP) as substrate. However, despite their high sequence similarity, they produced different diterpene products. While PtTPS19 formed exclusively ent-kaurene, PtTPS20 generated mainly the diterpene alcohol, 16α-hydroxy-ent-kaurane. Using homology-based structure modeling and site-directed mutagenesis, we demonstrated that one amino acid residue determines the different product specificity of PtTPS19 and PtTPS20. A reciprocal exchange of methionine 607 and threonine 607 in the active sites of PtTPS19 and PtTPS20, respectively, led to a complete interconversion of the enzyme product profiles. Gene expression analysis revealed that the diterpene synthase genes characterized showed organ-specific expression with the highest abundance of PtTPS17 and PtTPS20 transcripts in poplar roots. The poplar diterpene synthases PtTPS17, PtTPS19, and PtTPS20 contribute to the production of ent-kaurene and 16α-hydroxy-ent-kaurane in poplar. While ent-kaurene most likely serves as the universal precursor for gibberellins, the function of 16α-hydroxy-ent-kaurane in poplar is not known yet. However, the high expression levels of PtTPS20 and PtTPS17 in poplar roots may indicate an important function of 16α-hydroxy-ent-kaurane in secondary metabolism in this plant organ. The online version of this article (doi:10.1186/s12870-015-0647-6) contains supplementary material, which is available to authorized users.