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Abstract：
Homogentisate solanesyltransferase (HST) is a crucial enzyme in the plastoquinone biosynthetic pathway and has recently emerged as a promising target for herbicides. In this study, we successfully expressed and purified a stable and highly pure form of seven times transmembrane protein Chlamydomonas reinhardtii HST (CrHST). The final yield of CrHST protein obtained was 12.2 mg per liter of M9 medium. We evaluated the inhibitory effect on CrHST using Des-Morpholinocarbony Cyclopyrimorate (DMC) and found its IC50 value to be 3.63 ± 0.53 μM, indicating significant inhibitory potential. Additionally, we investigated the substrate affinity of CrHST with two substrates, determining the Km values as 22.76 ± 1.70 μM for FPP and 48.54 ± 3.89 μM for HGA. Through sequence alignment analyses and three-dimensional structure predictions, we identified conserved amino acid residues forming the active cavity in the enzyme. The results from molecular docking and binding energy calculations indicate that DMC has a greater binding affinity with HST compared to HGA. These findings represent substantial progress in understanding CrHST\'s properties and potential for herbicide development. KEY POINTS: • First high-yield transmembrane CrHST protein via E. coli system • Preliminarily identified active cavity composition via activity testing • Determined substrate and inhibitor modes via molecular docking.
摘要：
均质物茄二酰转移酶（HST）是质体醌生物合成途径中的关键酶，最近已成为除草剂的有希望的靶标。在这项研究中,我们成功表达并纯化了稳定且高纯度的七倍跨膜蛋白莱茵衣藻HST（CrHST）。获得的CrHST蛋白的最终产量为12.2mg/升M9培养基。我们使用Des-吗啉烯酸环吡啶（DMC）评估了对CrHST的抑制作用，发现其IC50值为3.63±0.53μM，表明显著的抑制潜力。此外,我们研究了CrHST与两种底物的底物亲和力，确定FPP的Km值为22.76±1.70μM，HGA的Km值为48.54±3.89μM。通过序列比对分析和三维结构预测,我们确定了在酶中形成活性腔的保守氨基酸残基。来自分子对接和结合能计算的结果表明,与HGA相比,DMC与HST具有更大的结合亲和力。这些发现代表了在理解CrHST的特性和除草剂开发潜力方面的实质性进展。关键点:•通过大肠杆菌系统的第一个高产跨膜CrHST蛋白•通过活性测试初步鉴定活性腔组成•通过分子对接确定底物和抑制剂模式。
