Zang Xin, Bat-Erdene Undramaa, Huang Weixue, Wu Zhongshou, Jacobsen Steve E, Tang Yi, Zhou Jiahai
Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA, USA.
Biodes Res. 2024 Nov 1;6:0046. doi: 10.34133/bdr.0046. eCollection 2024.
Dihydroxy acid dehydratase (DHAD) is the third enzyme in the plant branched-chain amino acid biosynthetic pathway and the target for commercial herbicide development. We have previously reported the discovery of fungal natural product aspterric acid (AA) as a submicromolar inhibitor of DHAD through self-resistance gene directed genome mining. Here, we reveal the mechanism of AA inhibition on DHAD and the self-resistance mechanism of AstD, which is encoded by the self-resistance gene D. As a competitive inhibitor, the hydroxycarboxylic acid group of AA mimics the binding of the natural substrate of DHAD, while the hydrophobic moiety of AA occupies the substrate entrance cavity. Compared to DHAD, AstD has a relatively narrow substrate channel to prevent AA from binding. Several mutants of DHAD were generated and assayed to validate the self-resistance mechanism and to confer DHAD with AA resistance. These results will lead to the engineering of new type of herbicides targeting DHAD and provide direction for the ecological construction of herbicide-resistant crops.
二羟基酸脱水酶（DHAD）是植物支链氨基酸生物合成途径中的第三种酶，也是商业除草剂开发的靶标。我们之前曾报道过，通过自抗性基因导向的基因组挖掘，发现真菌天然产物阿斯替酸（AA）是一种亚微摩尔级的DHAD抑制剂。在此，我们揭示了AA对DHAD的抑制机制以及由自抗性基因D编码的AstD的自我抗性机制。作为一种竞争性抑制剂，AA的羟基羧酸基团模拟了DHAD天然底物的结合，而AA的疏水部分占据了底物进入腔。与DHAD相比，AstD具有相对狭窄的底物通道，可防止AA结合。我们构建并检测了几种DHAD突变体，以验证自我抗性机制并赋予DHAD对AA的抗性。这些结果将推动新型靶向DHAD的除草剂的工程化，并为抗除草剂作物的生态建设提供方向。
