Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti-HIV meroterpenoid isolated from Rhododendron dauricum We identified a novel cDNA encoding DCA synthase by transcriptome-based screening from young leaves of R. dauricum The gene coded for a 533-amino acid polypeptide with moderate homologies to flavin adenine dinucleotide oxidases from other plants. The primary structure contained an amino-terminal signal peptide and conserved amino acid residues to form bicovalent linkage to the flavin adenine dinucleotide isoalloxazine ring at histidine-112 and cysteine-175. In addition, the recombinant DCA synthase, purified from the culture supernatant of transgenic Pichia pastoris, exhibited structural and functional properties as a flavoprotein. The reaction mechanism of DCA synthase characterized herein partly shares a similarity with those of cannabinoid synthases from Cannabis sativa, whereas DCA synthase catalyzes a novel cyclization reaction of the farnesyl moiety of a meroterpenoid natural product of plant origin. Moreover, in this study, we present evidence that DCA is biosynthesized and accumulated specifically in the glandular scales, on the surface of R. dauricum plants, based on various analytical studies at the chemical, biochemical, and molecular levels. The extracellular localization of DCA also was confirmed by a confocal microscopic analysis of its autofluorescence. These data highlight the unique feature of DCA: the final step of biosynthesis is completed in apoplastic space, and it is highly accumulated outside the scale cells.

中文翻译：

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抗 HIV 生物合成中活性的 Daurichromenic Acid Synthase 的鉴定和表征。


Daurichromenic Acid (DCA) 合酶催化灰叶酸氧化环化产生 DCA，一种从 Dauricum 杜鹃中分离出的抗 HIV 类萜 我们通过转录组筛选从 Dauricum 幼叶中鉴定出编码 DCA 合酶的新型 cDNA。一种 533 个氨基酸的多肽，与其他植物的黄素腺嘌呤二核苷酸氧化酶具有中等同源性。一级结构包含氨基末端信号肽和保守氨基酸残基，在组氨酸 112 和半胱氨酸 175 处与黄素腺嘌呤二核苷酸异咯嗪环形成双共价连接。此外，从转基因毕赤酵母培养物上清液中纯化的重组DCA合酶表现出黄素蛋白的结构和功能特性。本文表征的DCA合酶的反应机制部分地与来自大麻的大麻素合酶的反应机制相似，而DCA合酶催化植物来源的类萜天然产物的法呢基部分的新型环化反应。此外，在这项研究中，我们基于化学、生物化学和分子水平的各种分析研究，提供了证据表明 DCA 是专门在 R. dauricum 植物表面的腺鳞中生物合成和积累的。 DCA 的细胞外定位也通过其自发荧光的共聚焦显微镜分析得到证实。这些数据凸显了DCA的独特之处：生物合成的最后一步是在质外体空间完成的，并且在鳞片细胞外高度积累。