Identification and characterization of bacterial diterpene cyclases that synthesize the cembrane skeleton.
Identification and characterization of bacterial diterpene cyclases that synthesize the cembrane skeleton.
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DOI：
10.1002/cbic.201200651
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发表时间：
2013-02-11
期刊：
影响因子：
3.2
通讯作者：
Kuzuyama, Tomohisa
中科院分区：
文献类型：
--
作者：
Meguro, Ayuko;Tomita, Takeo;Nishiyama, Makoto;Kuzuyama, Tomohisa
萜类化合物，也称为类异戊二烯，是一个大的和高度多样化的天然产物组。[1-3]萜类化合物的结构多样性是有限数量的线性聚异戊二烯基二磷酸底物（例如香叶基二磷酸（GDP，C10）、法呢基二磷酸（FDP，C15）和香叶基香叶基二磷酸（GGDP，C20））环化以产生单环或多环化合物的结果。萜烯环化酶在萜类化合物的生物合成过程中起着关键作用，因为它们在涉及电离、氢化物移位、去质子化、质子化、甲基迁移和羟基化的反应级联中产生结构和立体化学上不同的环骨架。[1，3]萜烯环化酶反应级联是由高活性碳阳离子的形成引发的。这种碳阳离子的形成有两种机制：1）聚异戊二烯基二磷酸底物通过二磷酸提取的电离，和2）底物末端烯烃的质子化。[3-7]萜烯环化酶根据其机制分类。[3-7]I类萜烯环化酶通过底物电离启动碳阳离子形成，而II类萜烯环化酶通过底物质子化启动碳阳离子形成。I类萜烯脱氢酶含有DDXXD/E和/或NSE/DTE基序，其螯合用于结合底物的二磷酸部分并且是烯丙基二磷酸酯键电离所需的Mg 2+离子。相比之下，II类萜烯环化酶具有DXDD基序，其作为催化酸，使底物的末端碳碳双键质子化。注意，在由I类酶形成的反应产物中缺少二磷酸基团，而在由II类酶形成的反应产物中保留二磷酸基团。已知链霉菌属和其他放线菌的成员是革兰氏阳性细菌，它们产生大量的天然产物。然而，很少有二萜（C20）已从这些细菌中分离出来。已经从这些细菌中克隆了有限数量的与GGDP反应的二萜环化酶，并对其进行了表征，例如，来自产萜菌素的灰中北里孢菌的萜二烯基二磷酸合酶（Cyc1），[8，9]来自产viguiepinol的链霉菌属KO-3988的对映-柯巴基二磷酸合酶（SsCPS），[10，11]来自结核分枝杆菌H37的卤代二烯基二磷酸合酶（Rv3377c），[12]和环辛-9-烯-7-醇合酶（CotB2），来自产生环辛肽的产黑孢链霉菌MI614 - 43F2 [13]（支持信息中的图S1）。在这四种酶中，Cyc1、SsCPS和Rv3377c被认为是II类萜烯环化酶。如上所述，在由这些II类酶形成的反应产物中发现二磷酸基团。这些II类酶伴随有I类萜烯环化酶Cyc2，[8] ORF 3，[10]和Rv3378c，[14]，它们与II类萜烯环化酶（分别为Cyc1，SsCPS和Rv3377c）的每个反应产物反应以释放二磷酸基团（图S1）。有趣的是，Cyc1和SsCPS是通过它们与甲羟戊酸途径基因簇的接近而发现的，该基因簇提供了GGDP的异戊二烯单元前体。[8，10]然而，在上述四种酶中，CotB2被认为是I类二萜合酶，因为它具有NSE基序。[13]本文报道了从链霉菌SANK 60404中分离得到的两种新的二萜环化酶DtcycA和DtcycB，它们分别与CotB2和DtcycB的反应产物环辛-9-烯-7-醇（cyclooctat-9-en-7-ol）的反应产物环辛-9-烯-7-醇（cyclooctat-9-en-7-ol）的反应产物环辛-9-烯-7-醇（cyclooctat-7-ol）的反应产物环辛-9-烯-7-醇（cyclooctat-9-en-7-ol）的反应产物环辛-9-烯-7-醇（cyclooctat-7-ol）的反应产物环辛-9-烯-7-醇（cyclooctat-9-en-7-ol）。
Terpenoids, which are also known as isoprenoids, are a large and highly diverse group of natural products.[1–3] The structural diversity of terpenoids is a result of the cyclization of a limited number of linear polyprenyl diphosphate substrates, such as geranyl diphosphate (GDP, C10), farnesyl diphosphate (FDP, C15), and geranylgeranyl diphosphate (GGDP, C20), to generate monocyclic or multicyclic compounds. Terpene cyclases play a key role in the biosynthetic processes of terpenoids, because they yield structurally and stereochemically diverse ring skeletons in the reaction cascade, which involves ionization, hydride shifts, deprotonation, protonation, methyl migration, and hydroxylation.[1, 3] The terpene cyclase reaction cascade is initiated by the formation of a highly reactive carbocation. There are two mechanisms for this carbocation formation: 1) ionization of the polyprenyl diphosphate substrate by diphosphate abstraction, and 2) protonation of the terminal olefin of the substrate.[3–7] Terpene cyclases are classified according to their mechanisms.[3–7] Class I terpene synthases initiate carbocation formation by substrate ionization, whereas class II terpene cyclases initiate carbocation formation by substrate protonation. ClassI terpene synthases contain DDXXD/E and/or NSE/DTE motifs, which chelate the Mg2+ ions that are used to bind the diphosphate moiety of the substrate and are required for the ionization of the allylic diphosphate ester bond. The class II terpene cyclases, in contrast, possess a DXDD motif, which serves as the catalytic acid that protonates the terminal carbon–carbon double-bond of the substrate. Note that a diphosphate group is missing in the reaction product that is formed by class I enzymes, whereas a diphosphate group is retained in the reaction product formed by class II enzymes. Members of the Streptomyces genus and other actinomycetes, which are Gram-positive bacteria, are known to produce an enormous variety of natural products. However, few diterpenes (C20) have been isolated from these bacteria. A limited number of diterpene cyclases that react with GGDP having been cloned from these bacteria and characterized, for example, terpentedienyl diphosphate synthase (Cyc1) from terpentecin-producing Kitasatospora griseola,[8, 9] ent-copalyl diphosphate synthase (SsCPS) from viguiepinol-producing Streptomyces sp. KO-3988,[10, 11] halimadienyl diphosphate synthase (Rv3377c) from Mycobacterium tuberculosis H37,[12] and cyclooctat-9-en-7-ol synthase (CotB2) from cyclooctatin-producing Streptomyces melanosporofaciens MI614-43F2 [13](Figure S1 in the Supporting Information). Of these four enzymes, Cyc1, SsCPS, and Rv3377c are considered to be class II terpene cyclases. As mentioned above, a diphosphate group is found in the reaction product formed by these class II enzymes. These class II enzymes are accompanied by class I terpene synthases Cyc2,[8] ORF3,[10] and Rv3378c,[14] which react with each reaction product of the classII terpene cyclases (Cyc1, SsCPS, and Rv3377c, respectively) to release a diphosphate group (FigureS1). Interestingly, Cyc1 and SsCPS were found by their proximity to the mevalonate pathway gene cluster, which provides isoprene unit precursors of GGDP.[8, 10] However, of the four above-mentioned enzymes, CotB2 is considered to be a class I diterpene synthase because it possesses an NSE motif.[13] In addition, a diphosphate group is removed by the reaction catalyzed by CotB2 to form the reaction product cyclooctat-9-en-7-ol.Here, we report two novel diterpene cyclases, DtcycA and DtcycB, that were mined from Streptomyces sp. SANK 60404, which …
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影响因子：
3.2
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1.8
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