生物活性三萜具有复杂的稠环结构,主要由最初的酶形成,2,3-氧化角鲨烯环化酶(OSC)在植物三萜生物合成中的作用。三萜与B,C环开放的骨骼极其罕见,形成机制未知,藏有未知的化学和生物学。这里,通过挖掘藜麦的基因组,然后进行功能表征,我们确定了一种能够产生B的应激反应性和新功能化的OSC,C-环开三萜,包括作为专门的表皮膀胱细胞的蜡成分的山茶酚A和B以及新型的(-)-羟基喹啉A,即喹啉合酶(CqQS)。蛋白质结构分析,然后进行定点突变,确定了五环β-amyrin合酶(CqbAS1)和B之间功能性相互转换的关键可变氨基酸位点,C-开环三萜合酶CqQS。一个关键残基(N612K)的突变甚至在进化上遥远的拟南芥β-amyrin合酶可以产生喹啉,表明B的保守机制,植物中C-开环三萜的形成。量子计算与对接实验相结合进一步表明,CqQS的保守W613和F413的构象可能是选择性地稳定中间碳阳离子朝向B的关键,C-开环三萜的形成。我们的发现揭示了藜麦三萜骨骼多样性和B,C-开环三萜生物合成,为获得其化学和生物学开辟了途径,并为藜麦性状工程和质量改进铺平了道路。
Bioactive triterpenes feature complex fused-ring structures, primarily shaped by the first-committed enzyme, 2,3-oxidosqualene cyclases (OSCs) in plant triterpene biosynthesis. Triterpenes with B,C-ring-opened skeletons are extremely rare with unknown formation mechanisms, harbouring unchartered chemistry and biology. Here, through mining the genome of Chenopodium quinoa followed by functional characterization, we identified a stress-responsive and neofunctionalized OSC capable of generating B,C-ring-opened triterpenes, including camelliol A and B and the novel (-)-quinoxide A as wax components of the specialized epidermal bladder cells, namely the quinoxide synthase (CqQS). Protein structure analysis followed by site-directed mutagenesis identified key variable amino acid sites underlying functional interconversion between pentacyclic β-amyrin synthase (CqbAS1) and B,C-ring-opened triterpene synthase CqQS. Mutation of one key residue (N612K) in even evolutionarily distant Arabidopsis β-amyrin synthase could generate quinoxides, indicating a conserved mechanism for B,C-ring-opened triterpene formation in plants. Quantum computation combined with docking experiments further suggests that conformations of conserved W613 and F413 of CqQS might be key to selectively stabilizing intermediate carbocations towards B,C-ring-opened triterpene formation. Our findings shed light on quinoa triterpene skeletal diversity and mechanisms underlying B,C-ring-opened triterpene biosynthesis, opening avenues towards accessing their chemistry and biology and paving the way for quinoa trait engineering and quality improvement.