ent-Kaurene is a biosynthetic intermediate diterpene of phytohormone gibberellins, and is biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP). The successive cyclization is catalyzed by two distinct diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS). Homologs of these diterpene synthase genes have been reported to be involved in the biosynthesis of specialized-metabolic diterpenoids for defense in several plant species, including rice (Oryza sativa). These diterpene synthases consist of three domains, αβγ domains. Active sites of ent-CPS exist at the interface of β and γ domain, while those of KS are located within the α domain. We herein carried out domain-deletion experiments using several KSs and KS like enzymes (KSLs) to obtain insights into the roles of domains other than active-site domains. As previously reported in taxadiene synthase, deletion of γ or βγ domains drastically decreased activities of specialized-metabolic OsKSL5, OsKSL8, OsKSL7 and OsKSL10 in O. sativa. However, unexpectedly, only α domains of several gibberellin-biosynthetic KSs, including OsKS1 in O. sativa, AtKS in Arabidopsis thaliana, TaKS in wheat (Triticum aestivum) and BdKS1 in Brachypodium distachyon, retained their original functions. Additionally, the specialized-metabolic OsKSL4, which is closely related to OsKS1, also functioned without its βγ domains. Domain-swapping experiments showed that replacing βγ domains in OsKSL7 with those from other KS/KSLs retained the OsKSL7 activity. Moreover, deletion of βγ domains of bifunctional PpCPS/KS in moss (Physcomitrella patens) drastically impaired its KS-related activity. Thus, we demonstrate that monofunctional gibberellin-biosynthetic KSs are the unique diterpene synthases that retain their functions without βγ domains.

中文翻译：

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高等植物中的赤霉素生物合成对映贝壳杉烯合酶不需要其非催化结构域进行催化


ent-贝壳杉烯是植物激素赤霉素的生物合成中间体二萜，由香叶基香叶基二磷酸通过对映柯巴基二磷酸（ent-CDP）生物合成。连续环化由两种不同的二萜合酶、ent-CDP 合酶 (ent-CPS) 和 ent-贝壳杉烯合酶 (KS) 催化。据报道，这些二萜合酶基因的同源物参与了包括水稻（Oryza sativa）在内的多种植物物种中用于防御的专门代谢二萜类化合物的生物合成。这些二萜合酶由三个结构域组成，即 αβγ 结构域。 ent-CPS的活性位点位于β和γ结构域的界面处，而KS的活性位点位于α结构域内。我们在此使用几种 KS 和 KS 样酶 (KSL) 进行结构域删除实验，以深入了解活性位点结构域以外的结构域的作用。正如之前在紫杉二烯合酶中报道的那样，γ 或 βγ 结构域的缺失大大降低了水稻中专门代谢的 OsKSL5、OsKSL8、OsKSL7 和 OsKSL10 的活性。然而，出乎意料的是，几种赤霉素生物合成KS，包括水稻中的OsKS1、拟南芥中的AtKS、小麦中的TaKS和二穗短柄草中的BdKS1，只有α结构域保留了其原始功能。此外，与 OsKS1 密切相关的专门代谢 OsKSL4 在没有 βγ 结构域的情况下也能发挥作用。结构域交换实验表明，用其他 KS/KSL 的结构域替换 OsKSL7 中的 βγ 结构域保留了 OsKSL7 的活性。此外，苔藓（Physcomitrella patens）中双功能 PpCPS/KS 的 βγ 结构域的缺失会严重损害其 KS 相关活性。 因此，我们证明单功能赤霉素生物合成 KS 是独特的二萜合酶，在没有 βγ 结构域的情况下仍保留其功能。