Li Tianfu, Shu Dan, Lei Lei, Li Zhemin, Luo Di, Yang Jie, Wang Yifan, Hou Xiaonan, Wang Hongning, Tan Hong
CAS Key Laboratory of Environmental and Applied Microbiology, Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China.
Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, China.
J Agric Food Chem. 2025 Jan 8;73(1):835-846. doi: 10.1021/acs.jafc.4c07116. Epub 2024 Dec 17.
BcABA3 is an unusual sesquiterpene synthase that lacks the conserved DDxxD and DTE/NSE motifs. Despite this, it can catalyze the conversion of farnesyl diphosphate to 2Z,4E-α-ionylideneethane. We used structure prediction, multiscale simulations, and site-directed mutagenesis experiments to investigate BcABA3 and its catalytic mechanism. BcABA3 has structural similarity to typical class I terpenoid cyclases in its active site. Based on simulation results, we identified two discontinuous glutamate residues, E124 and E88, which compensate for the absence of the aspartate-rich DDxxD motif. Quantum chemical calculations show that BcABA3 adopts a direct rotation mechanism for allyl cation isomerization rather than via the nerolidyl diphosphate. Then, it can achieve a successive proton transfer reaction, which is difficult to achieve by intramolecular rearrangement via the protruding outward carbonyl oxygen of A206. This reaction is then directed forward by two relatively stable intermediates containing a cation-conjugated double-bond structure. E124 is also proposed as the proton receptor in the final deprotonation to couple this step with 2Z,4E-α-ionylideneethane release. These findings provide valuable insight into the catalytic mechanisms of BcABA3 and can aid in its engineering, which will facilitate studies of abscisic acid biosynthesis.
BcABA3是一种不同寻常的倍半萜合酶，它缺乏保守的DDxxD和DTE/NSE基序。尽管如此，它仍能催化法呢基二磷酸转化为2Z,4E-α-紫罗烯亚基乙烷。我们利用结构预测、多尺度模拟和定点诱变实验来研究BcABA3及其催化机制。BcABA3在其活性位点与典型的I类萜类环化酶具有结构相似性。基于模拟结果，我们鉴定出两个不连续的谷氨酸残基E124和E88，它们弥补了富含天冬氨酸的DDxxD基序的缺失。量子化学计算表明，BcABA3采用直接旋转机制进行烯丙基阳离子异构化，而不是通过橙花叔醇二磷酸。然后，它可以实现连续的质子转移反应，这是通过A206向外突出的羰基氧进行分子内重排难以实现的。该反应随后由两个含有阳离子共轭双键结构的相对稳定的中间体引导向前。E124也被认为是最终去质子化中的质子受体，以使这一步骤与2Z,4E-α-紫罗烯亚基乙烷的释放相耦合。这些发现为BcABA3的催化机制提供了有价值的见解，并有助于其工程改造，这将促进脱落酸生物合成的研究。
