Department of Botany, Savitribai Phule Pune University, Pune, 411007, India.
Department of Microbiology, Savitribai Phule Pune University, Pune, 411007, India.
Planta. 2022 Oct 25;256(6):102. doi: 10.1007/s00425-022-04014-x.
Novel cytochrome P450s, CYP81B140 and CYP81B141 from Plumbago zeylanica were functionally characterized to understand their involvement in polyketide plumbagin biosynthesis. Further, we propose 3-methyl-1-8-naphthalenediol and isoshinanolone as intermediates for plumbagin biosynthesis. Plumbago zeylanica L. (P. zeylanica) is a medicinally important plant belonging to the family Plumbaginaceae. It comprises the most abundant naphthoquinone plumbagin having anti-cancer activity. Only the polyketide synthase (PKS) enzyme has been identified from the biosynthetic pathway which catalyzes iterative condensation of acetyl-CoA and malonyl-CoA molecules. The plumbagin biosynthesis involves hydroxylation, oxidation, hydration and dehydration of intermediate compounds which are expected to be catalyzed by cytochrome P450s (CYPs). To identify the CYPs, co-expression analysis was carried out using PKS as a candidate gene. Out of the eight identified CYPs, CYP81B140 and CYP81B141 have similar expression with PKS and belong to the CYP81 family. Phylogenetic analysis suggested that CYP81B140 and CYP81B141 cluster with CYPs from CYP81B, CYP81D, CYP81E and CYP81AA subfamilies which are known to be involved in the hydroxylation and oxidation reactions. Moreover, artificial microRNA-mediated transient individual silencing and co-silencing of CYP81B140 and CYP81B141 significantly reduced plumbagin and increased the 3-methyl-1-8-naphthalenediol and isoshinanolone content. Based on metabolite analysis, we proposed that 3-methyl-1-8-naphthalenediol and isoshinanolone function as intermediates for plumbagin biosynthesis. Transient silencing, over-expression and docking analysis revealed that CYP81B140 is involved in C-1 oxidation, C-4 hydroxylation and [C2-C3] hydration of 3-methyl-1-8-naphthalenediol to form isoshinanolone, whereas CYP81B141 is catalyzing [C2-C3] dehydration and C-4 oxidation of isoshinanolone to form plumbagin. Our results indicated that both CYP81B140 and CYP81B141 are promiscuous and necessary for plumbagin biosynthesis. This is the first report of identification and functional characterization of P. zeylanica-specific CYPs involved in plumbagin biosynthetic pathway and in general hexaketide synthesis in plants.
从白花丹中分离得到的新型细胞色素 P450s，CYP81B140 和 CYP81B141，其功能被鉴定以了解它们在聚酮普罗布醌生物合成中的作用。此外，我们提出 3-甲基-1-8-萘二醇和异羟基安息香酮作为普罗布醌生物合成的中间体。白花丹（Plumbago zeylanica）是属于白花丹科的一种药用植物。它含有最丰富的具有抗癌活性的萘醌普罗布醌。生物合成途径中只鉴定到聚酮合酶（PKS）酶，该酶催化乙酰辅酶 A 和丙二酰辅酶 A 分子的反复缩合。普罗布醌生物合成涉及中间化合物的羟化、氧化、水合和脱水，预计这些反应由细胞色素 P450s（CYPs）催化。为了鉴定 CYPs，我们使用 PKS 作为候选基因进行了共表达分析。在鉴定出的 8 个 CYP 中，CYP81B140 和 CYP81B141 与 PKS 的表达相似，属于 CYP81 家族。系统发育分析表明，CYP81B140 和 CYP81B141 与 CYP81B、CYP81D、CYP81E 和 CYP81AA 亚家族的 CYP 聚类，这些 CYP 已知参与羟化和氧化反应。此外，人工 microRNA 介导的瞬时个体沉默和 CYP81B140 和 CYP81B141 的共沉默显著降低了普罗布醌的含量，增加了 3-甲基-1-8-萘二醇和异羟基安息香酮的含量。基于代谢物分析，我们提出 3-甲基-1-8-萘二醇和异羟基安息香酮是普罗布醌生物合成的中间体。瞬时沉默、过表达和对接分析表明，CYP81B140 参与 3-甲基-1-8-萘二醇的 C-1 氧化、C-4 羟化和 [C2-C3] 水合，形成异羟基安息香酮，而 CYP81B141 催化异羟基安息香酮的 [C2-C3] 脱水和 C-4 氧化，形成普罗布醌。我们的结果表明，CYP81B140 和 CYP81B141 都是普罗布醌生物合成途径和植物中一般六酮合成所必需的混杂酶。这是首次报道参与普罗布醌生物合成途径和植物中一般六酮合成的白花丹特异性 CYP 的鉴定和功能特征。
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