Mutational analysis of white spruce (Picea glauca) ent-kaurene synthase (PgKS) reveals common and distinct mechanisms of conifer diterpene synthases of general and specialized metabolism
Mutational analysis of white spruce (Picea glauca) ent-kaurene synthase (PgKS) reveals common and distinct mechanisms of conifer diterpene synthases of general and specialized metabolism
复制标题
DOI：
10.1016/j.phytochem.2011.11.004
复制
发表时间：
2012-02-01
期刊：
影响因子：
3.8
通讯作者：
Bohlmann, Joerg
中科院分区：
文献类型：
--
作者：
Zerbe, Philipp;Chiang, Angela;Bohlmann, Joerg
针叶树二萜脱氢酶（diTPS）催化香叶基香叶基二磷酸或柯巴基二磷酸的多步环异构化，通常为多种二萜（即，主要的）和专门的（即，次级）代谢。尽管它们的功能多样性，已知的针叶树diTPS在结构上密切相关，在三个保守的结构域，α，β和γ的变化。针叶树I类和I/II类diTPS的催化特异性主要由C-末端I类活性位点的蛋白质环境通过稳定常见和独特的碳阳离子中间体来确定。以短叶红豆杉（Taxus brevifolia）紫杉二烯合酶（Taxadiene synthase，TSAs）的晶体结构为模板，对白云杉（Picea glauca）中的第一类diTPS对映贝壳杉烯合酶（PgKS）进行了比较建模和诱变，以阐明PgKS相对于专门代谢的云杉diTPS的催化特异性。N-末端截短证明了PgKS的I类酶活性中By结构域的作用，促进了底物结合后I类活性位点的关闭。基于位置，Arg 476和Asp 736在C-末端的PgKS结构域可能有助于这种构象转变，并出现催化的关键。与其他diTPS的机制一致，柯巴基二磷酸底物的后续电离和二磷酸基团的配位由PgKS的DDxxD和NDIQGCKRE基序中严格保守的残基控制，例如Asn 656和Arg 653。此外，Lys 478、Trp 502、Met 588、Ala 615和Ile 619通过碳阳离子稳定化来控制PgKS的酶活性和特异性。这些位置显示出高水平的氨基酸变异，与一般或专门代谢中不同功能的针叶树diTPS之间的功能可塑性一致。(C)2011爱思唯尔有限公司版权所有。
Conifer diterpene synthases (diTPSs) catalyze the multi-step cycloisomerization of geranylgeranyl diphosphate, or copalyl diphosphate, to a variety of diterpenes in general (i.e., primary) and specialized (i.e., secondary) metabolism. Despite their functional diversity, the known conifer diTPSs are structurally closely related, with variations in three conserved domains, alpha, beta and gamma. The catalytic specificity of conifer class I and class I/II diTPSs is predominantly determined by the protein environment of the C-terminal class I active site through stabilization of common and unique carbocation intermediates. Using the crystal structure of Taxus brevifolia taxadiene synthase as template, comparative modeling and mutagenesis of the class I diTPS ent-kaurene synthase from Picea glauca (PgKS) was performed to elucidate the catalytic specificity of PgKS relative to spruce diTPSs of specialized metabolism. N-terminal truncations demonstrated a role for the By domain in class I enzyme activity for PgKS, facilitating the closure of the class I active site upon substrate binding. Based on position, Arg476 and Asp736 in the C-terminal a domain of PgKS may contribute to this conformational transition and appear critical for catalysis. Consistent with the mechanism of other diTPSs, the subsequent ionization of a copalyl diphosphate substrate and coordination of the diphosphate group is controlled by strictly conserved residues in the DDxxD and NDIQGCKRE motif of PgKS, such as Asn656 and Arg653. Furthermore, Lys478, Trp502, Met588, Ala615 and Ile619 control the enzymatic activity and specificity of PgKS via carbocation stabilization en route to ent-kaurene. These positions show a high level of amino acid variation, consistent with functional plasticity among conifer diTPSs of different functions in general or specialized metabolism. (C) 2011 Elsevier Ltd. All rights reserved.