Abe Tohru, Shiratori Haruna, Kashiwazaki Kosuke, Hiasa Kazuma, Ueda Daijiro, Taniguchi Tohru, Sato Hajime, Abe Takashi, Sato Tsutomu
Department of Life and Food Sciences, Graduate School of Science and Technology, Niigata University Ikarashi 2-8050, Nishi-ku Niigata 950-2181 Japan
Department of Electrical and Information Engineering, Graduate School of Science and Technology, Niigata University Ikarashi 2-8050, Nishi-ku Niigata 950-2181 Japan
Chem Sci. 2024 Jun 5;15(27):10402-10407. doi: 10.1039/d4sc01381f. eCollection 2024 Jul 10.
Non-canonical terpene synthases (TPSs) with primary sequences that are unrecognizable as canonical TPSs have evaded detection by conventional genome mining. This study aimed to prove that novel non-canonical TPSs can be efficiently discovered from proteins, hidden in genome databases, predicted to have 3D structures similar to those of class I TPSs. Six types of non-canonical TPS candidates were detected using this search strategy from 268 genome sequences from actinomycetes. Functional analyses of these candidates revealed that at least three types were novel non-canonical TPSs. We propose classifying the non-canonical TPSs as classes ID, IE, and IF. A hypothetical protein MBB6373681 from (PeuTPS) was selected as a representative example of class ID TPSs and characterized. PeuTPS was identified as a diterpene synthase that forms a 6/6/6-fused tricyclic gersemiane skeleton. Analyses of PeuTPS variants revealed that amino acid residues within new motifs [D(N/D), ND, and RXXKD] located close to the class I active site in the 3D structure were essential for enzymatic activity. The homologs of non-canonical TPSs found in this study exist in bacteria as well as in fungi, protists, and plants, and the PeuTPS gene is not located near terpene biosynthetic genes in the genome. Therefore, structural-model-based genome mining is an efficient strategy to search for novel non-canonical TPSs that are independent of biological species and biosynthetic gene clusters and will contribute to expanding the structural diversity of terpenoids.
具有无法被识别为典型萜类合酶（TPSs）的一级序列的非典型萜类合酶，一直未被传统的基因组挖掘检测到。本研究旨在证明，可以从隐藏在基因组数据库中的蛋白质中高效发现新型非典型TPSs，这些蛋白质被预测具有与I类TPSs相似的三维结构。使用这种搜索策略，从268个放线菌基因组序列中检测到六种非典型TPS候选物。对这些候选物的功能分析表明，至少有三种类型是新型非典型TPSs。我们建议将非典型TPSs分类为ID、IE和IF类。从 （PeuTPS）中选择的一个假定蛋白MBB6373681作为ID类TPSs的代表性实例并进行了表征。PeuTPS被鉴定为一种形成6/6/6稠合三环格氏半萜骨架的二萜合酶。对PeuTPS变体的分析表明，在三维结构中靠近I类活性位点的新基序[D（N/D）、ND和RXXKD]内的氨基酸残基对酶活性至关重要。本研究中发现的非典型TPSs的同源物存在于细菌以及真菌、原生生物和植物中，并且PeuTPS基因在基因组中并不位于萜类生物合成基因附近。因此，基于结构模型的基因组挖掘是一种搜索新型非典型TPSs的有效策略，该策略独立于生物物种和生物合成基因簇，将有助于扩大萜类化合物的结构多样性。
