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Biochemical characterization of microbial type terpene synthases in two closely related species of hornworts, Anthoceros punctatus and Anthoceros agrestis
Phytochemistry ( IF 3.2 ) Pub Date : 2018-05-01 , DOI: 10.1016/j.phytochem.2018.02.011
Wangdan Xiong , Jianyu Fu , Tobias G. Köllner , Xinlu Chen , Qidong Jia , Haobo Guo , Ping Qian , Hong Guo , Guojiang Wu , Feng Chen

Microbial terpene synthase-like (MTPSL) genes are a type of terpene synthase genes only recently identified in plants. In contrast to typical plant terpene synthase genes, which are ubiquitous in land plants, MTPSL genes appear to occur only in nonseed plants. Our knowledge of catalytic functions of MTPSLs is very limited. Here we report biochemical characterization of the enzymes encoded by MTPSL genes from two closely related species of hornworts, Anthoceros punctatus and Anthoceros agrestis. Seven full-length MTPSL genes were identified in A. punctatus (ApMTPSL1-7) based on the analysis of its genome sequence. Using homology-based cloning, the apparent orthologs for six of the ApMTPSL genes, except ApMTPSL2, were cloned from A. agrestis. They were designated AaMTPSL1, 3-7. The coding sequences for each of the 13 Anthoceros MTPSL genes were cloned into a protein expression vector. Escherichia coli-expressed recombinant MTPSLs from hornworts were assayed for terpene synthase activities. Six ApMTPSLs and five AaMTPSLs, except for ApMTPSL5 and AaMTPSL5, showed catalytic activities with one or more isoprenyl diphosphate substrates. All functional MTPSLs exhibited sesquiterpene synthase activities. In contrast, only ApMTPSL7 and AaMTPSL7 showed monoterpene synthase activity and only ApMTPSL2, ApMTPSL6 and AaMTPSL6 showed diterpene synthase activity. Most MTPSLs from Anthoceros contain uncanonical aspartate-rich motif in the form of either 'DDxxxD' or 'DDxxx'. Homology-based structural modeling analysis of ApMTPSL1 and ApMTPSL7, which contain 'DDxxxD' and 'DDxxx' motif, respectively, showed that 'DDxxxD' and 'DDxxx' motifs are localized in the similar positions as the canonical 'DDxxD' motif in known terpene synthases. To further understand the role of individual aspartate residues in the motifs, ApMTPSL1 and ApMTPSL7 were selected as two representatives for site-directed mutagenesis studies. No activities were detected when any of the conserved aspartic acid was mutated into alanine. This study provides new information about the catalytic functions of MTPSLs and the functionality of their uncanonical aspartate-rich motifs, and builds a knowledge base for studying the biological importance of MTPSL genes and their terpene products in nonseed plants.

中文翻译:

两种密切相关的金鱼属植物 Anhoceros punctatus 和 Anhoceros agrestis 中微生物型萜烯合酶的生化特征

微生物萜烯合酶样 (MTPSL) 基因是最近才在植物中发现的一种萜烯合酶基因。与在陆地植物中普遍存在的典型植物萜烯合酶基因相比,MTPSL 基因似乎只出现在非种子植物中。我们对 MTPSL 催化功能的了解非常有限。在这里,我们报告了由来自两种密切相关的金鱼属植物、Anhoceros punctatus 和 Anhoceros agrestis 的 MTPSL 基因编码的酶的生化特征。基于对 A. punctatus (ApMTPSL1-7) 基因组序列的分析,在 A. punctatus (ApMTPSL1-7) 中鉴定了七个全长 MTPSL 基因。使用基于同源性的克隆,从 A.agrestis 克隆了六个 ApMTPSL 基因(ApMTPSL2 除外)的表观直系同源物。它们被指定为 AaMTPSL1, 3-7。将 13 个 Anthoceros MTPSL 基因中的每一个的编码序列克隆到蛋白质表达载体中。分析了大肠杆菌表达的来自金鱼草的重组 MTPSL 的萜烯合酶活性。除了 ApMTPSL5 和 AaMTPSL5 之外,6 个 ApMTPSL 和 5 个 AaMTPSL 显示出对一种或多种异戊二烯二磷酸底物的催化活性。所有功能性 MTPSL 均表现出倍半萜烯合酶活性。相比之下,只有 ApMTPSL7 和 AaMTPSL7 显示出单萜合酶活性,只有 ApMTPSL2、ApMTPSL6 和 AaMTPSL6 显示出二萜合酶活性。来自 Anthoceros 的大多数 MTPSL 都包含“DDxxxD”或“DDxxx”形式的不规范的富含天冬氨酸的基序。ApMTPSL1 和 ApMTPSL7 的基于同源性的结构建模分析,它们分别包含“DDxxxD”和“DDxxx”基序,表明“DDxxxD”和“DDxxx”基序定位在与已知萜合酶中的典型“DDxxD”基序相似的位置。为了进一步了解单个天冬氨酸残基在基序中的作用,选择 ApMTPSL1 和 ApMTPSL7 作为定点诱变研究的两个代表。当任何保守的天冬氨酸突变为丙氨酸时,未检测到活性。本研究提供了有关 MTPSL 的催化功能及其非典型富含天冬氨酸基序功能的新信息,并为研究 MTPSL 基因及其萜烯产物在非种子植物中的生物学重要性奠定了知识基础。为了进一步了解单个天冬氨酸残基在基序中的作用,选择 ApMTPSL1 和 ApMTPSL7 作为定点诱变研究的两个代表。当任何保守的天冬氨酸突变为丙氨酸时,未检测到活性。本研究提供了有关 MTPSL 的催化功能及其非典型富含天冬氨酸基序功能的新信息,并为研究 MTPSL 基因及其萜烯产物在非种子植物中的生物学重要性奠定了知识基础。为了进一步了解单个天冬氨酸残基在基序中的作用,选择 ApMTPSL1 和 ApMTPSL7 作为定点诱变研究的两个代表。当任何保守的天冬氨酸突变为丙氨酸时,未检测到活性。本研究提供了有关 MTPSL 的催化功能及其非典型富含天冬氨酸基序功能的新信息,并为研究 MTPSL 基因及其萜烯产物在非种子植物中的生物学重要性奠定了知识基础。
更新日期:2018-05-01
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