T Rehka, Sharma Deepti, Lin Fu, Choong Yeu Khai, Lim Chinchin, Jobichen Chacko, Zhang Congqiang
Singapore Institute of Food and Biotechnology Innovation (SIFBI), Agency for Science, Technology and Research (ASTAR), Singapore 138669, Singapore.
CNRS@CREATE, Singapore 138602, Singapore.
ACS Catal. 2023 Mar 27;13(7):4949-4959. doi: 10.1021/acscatal.2c05598. eCollection 2023 Apr 7.
Terpene synthases (TPSs), known gatekeepers of terpenoid diversity, are the main targets for enzyme engineering attempts. To this end, we have determined the crystal structure of linalool synthase (Ap.LS), which has been recently reported to be 44-fold and 287-fold more efficient than bacterial and plant counterparts, respectively. Structure-based molecular modeling followed by as well as tests confirmed that the region of 60-69aa and Tyr299 (adjacent to the motif "WxxxxxRY") are essential for maintaining Ap.LS specificity toward a short-chain (C10) acyclic product. Ap.LS Y299 mutants (Y299A, Y299C, Y299G, Y299Q, and Y299S) yielded long-chain (C15) linear or cyclic products. Molecular modeling based on the Ap.LS crystal structure indicated that farnesyl pyrophosphate in the binding pocket of Ap.LS Y299A has less torsion strain energy compared to the wild-type Ap.LS, which can be partially attributed to the larger space in Ap.LS Y299A for better accommodation of the longer chain (C15). Linalool/nerolidol synthase Y298 and humulene synthase Y302 mutations also produced C15 cyclic products similar to Ap.LS Y299 mutants. Beyond the three enzymes, our analysis confirmed that most microbial TPSs have asparagine at the position and produce mainly cyclized products (δ-cadinene, 1,8-cineole, epi-cubebol, germacrene D, β-barbatene, etc.). In contrast, those producing linear products (linalool and nerolidol) typically have a bulky tyrosine. The structural and functional analysis of an exceptionally selective linalool synthase, Ap.LS, presented in this work provides insights into factors that govern chain length (C10 or C15), water incorporation, and cyclization (cyclic vs acyclic) of terpenoid biosynthesis.
萜类合酶（TPSs）是萜类多样性的已知守门人，是酶工程尝试的主要目标。为此，我们确定了芳樟醇合酶（Ap.LS）的晶体结构，最近有报道称其效率分别比细菌和植物中的对应物高44倍和287倍。基于结构的分子建模以及测试证实，60 - 69aa区域和Tyr299（与基序“WxxxxxRY”相邻）对于维持Ap.LS对短链（C10）无环产物的特异性至关重要。Ap.LS Y299突变体（Y299A、Y299C、Y299G、Y299Q和Y299S）产生长链（C15）线性或环状产物。基于Ap.LS晶体结构的分子建模表明，与野生型Ap.LS相比，Ap.LS Y299A结合口袋中的法呢基焦磷酸具有更低的扭转应变能，这部分归因于Ap.LS Y299A中更大的空间，以便更好地容纳更长的链（C15）。芳樟醇/橙花叔醇合酶Y298和葎草烯合酶Y302突变也产生了与Ap.LS Y299突变体类似的C15环状产物。除了这三种酶，我们的分析证实，大多数微生物TPSs在该位置具有天冬酰胺，并主要产生环化产物（δ-杜松烯、1,8-桉叶素、表古巴醇、吉马烯D、β-巴巴烯等）。相反，那些产生线性产物（芳樟醇和橙花叔醇）的通常具有一个大体积的酪氨酸。这项工作中对一种具有异常选择性的芳樟醇合酶Ap.LS的结构和功能分析，为萜类生物合成中控制链长（C10或C15）、水的掺入以及环化（环状与无环）的因素提供了见解。
