Bifunctional abletadiene synthase: Mutual structural dependence of the active sites for protonation-initiated and ionization-initiated cyclizations
Bifunctional abletadiene synthase: Mutual structural dependence of the active sites for protonation-initiated and ionization-initiated cyclizations
复制标题
DOI：
10.1021/bi020492n
复制
发表时间：
2003-03-11
期刊：
影响因子：
2.9
通讯作者：
Croteau, RB
中科院分区：
文献类型：
--
作者：
Peters, RJ;Carter, OA;Croteau, RB
来自大冷杉的松香二烯合酶催化香叶基香叶基二磷酸和柯巴基二磷酸的两个连续的、机械上不同的环化，形成松香二烯异构体的混合物，作为二萜类树脂酸生物合成的关键步骤。每个反应都是在一个单独的活性位点独立进行的，该活性位点位于被认为是萜烯环化酶典型的结构不同的域中。尽管存在不寻常的 250 个残基 N 端插入元件，但插入远端的一对串联带电残基被证明形成了 C 端活性位点的功能部分。由于松香二烯合酶类似于祖先植物萜烯环化酶，因此这一观察结果表明，此类酶的 N 末端具有重要的催化作用的带正电荷残基的早期进化起源。构建并表征了该酶的一系列 N 端和 C 端截断，无论是单独的还是作为相邻多肽对的混合物，以评估所提出的结构域结构、插入元件的功能以及假定的结构域间接触的作用。这些研究表明功能折叠中需要插入元件，并允许定义 N 端和 C 端活性位点肽的最小一级结构。最重要的是，结果表明，尽管松香二烯合酶的两个活性位点在催化上是独立的，但两个区域之间的实质性接触对于该酶的功能能力至关重要。因此，松香二烯合酶的两个环化位点不能被分割成催化上不同的结构域，因此松香二烯合酶不太可能是通过两个先前独立的基因的融合而产生的。
Abietadiene synthase from grand fir catalyzes two sequential, mechanistically distinct cyclizations, of geranylgeranyl diphosphate and of copalyl diphosphate, in the formation of a mixture of abietadiene isomers as the committed step of diterpenoid resin acid biosynthesis. Each reaction is independently conducted at a separate active site residing in what were considered to be structurally distinct domains typical of terpene cyclases. Despite the presence of an unusual 250-residue N-terminal insertional element, a tandem pair of charged residues distal to the insertion was shown to form a functional part of the C-terminal active site. Because abietadiene synthase resembles the ancestral plant terpene cyclase, this observation suggests an early evolutionary origin of catalytically important positively charged residues at the N-terminus of enzymes of this general class. A series of N- and C-terminal truncations of this enzyme were constructed and characterized, both alone and as mixtures of adjacent polypeptide pairs, to assess the proposed domain architecture, the function of the insertional element, and the role of presumptive interdomain contacts. These studies indicated a requirement for the insertional element in functional folding and allowed definition of the minimum primary structure of N- and C-terminal active site peptides. Most importantly, the results showed that, although the two active sites of abietadiene synthase are catalytically independent, substantial contact between the two regions is essential for the functional competence of this enzyme. Thus, the two cyclization sites of abietadiene synthase cannot be dissected into catalytically distinct domains, and, therefore, abietadiene synthase is unlikely to have arisen by fusion of two previously independent genes.