Characterization of a Flavoprotein Oxidase from Opium Poppy Catalyzing the Final Steps in Sanguinarine and Papaverine Biosynthesis
Characterization of a Flavoprotein Oxidase from Opium Poppy Catalyzing the Final Steps in Sanguinarine and Papaverine Biosynthesis
复制标题
DOI：
10.1074/jbc.m112.420414
复制
发表时间：
2012-12-14
影响因子：
4.8
通讯作者：
Facchini, Peter J.
中科院分区：
文献类型：
--
作者：
Hagel, Jillian M.;Beaudoin, Guillaume A. W.;Facchini, Peter J.
苄基异喹啉生物碱是一类植物特化代谢产物，包括镇痛吗啡、抗菌药血根碱和小檗碱以及血管扩张剂罂粟碱。二氢苯并菲啶氧化酶（DBOX）催化的二氢血根碱双电子氧化反应是血根碱生物合成的最后一步。血根碱中完全共轭环系的形成类似于（S）-坎那丁氧化成小檗碱和（S）-四氢罂粟碱氧化成罂粟碱的四电子氧化反应。我们报告的分离和功能特性的罂粟（罂粟）cDNA编码DBOX，黄素蛋白氧化酶与同源性（S）-四氢原小檗碱氧化酶和小檗碱桥酶。使用小檗碱桥酶查询翻译的罂粟茎转录组数据库产生了几个候选基因，包括选择用于在巴斯德毕赤酵母中异源表达的（S）-四氢原小檗碱氧化酶样序列。重组酶优先催化二氢血根碱氧化为血根碱，但也将（RS）-四氢罂粟碱转化为罂粟碱，并将几种原小檗碱生物碱转化为氧化形式，包括将（RS）-坎那丁转化为小檗碱。二氢血根碱和原小檗碱生物碱（S）-scoulavine的Km值分别为201和146 μ M，表明这些底物在植物中的浓度高。病毒诱导的基因沉默，以减少DBOX转录水平导致相应的减少血根碱，二氢血根碱，罂粟碱积累在支持DBOX作为一个多功能氧化酶在BIA代谢。
Benzylisoquinoline alkaloids are a diverse class of plant specialized metabolites that includes the analgesic morphine, the antimicrobials sanguinarine and berberine, and the vasodilator papaverine. The two-electron oxidation of dihydrosanguinarine catalyzed by dihydrobenzophenanthridine oxidase (DBOX) is the final step in sanguinarine biosynthesis. The formation of the fully conjugated ring system in sanguinarine is similar to the four-electron oxidations of (S)-canadine to berberine and (S)-tetrahydropapaverine to papaverine. We report the isolation and functional characterization of an opium poppy (Papaver somniferum) cDNA encoding DBOX, a flavoprotein oxidase with homology to (S)-tetrahydroprotoberberine oxidase and the berberine bridge enzyme. A query of translated opium poppy stem transcriptome databases using berberine bridge enzyme yielded several candidate genes, including an (S)-tetrahydroprotoberberine oxidase-like sequence selected for heterologous expression in Pichia pastoris. The recombinant enzyme preferentially catalyzed the oxidation of dihydrosanguinarine to sanguinarine but also converted (RS)-tetrahydropapaverine to papaverine and several protoberberine alkaloids to oxidized forms, including (RS)-canadine to berberine. The Km values of 201 and 146 mu M for dihydrosanguinarine and the protoberberine alkaloid (S)-scoulerine, respectively, suggested high concentrations of these substrates in the plant. Virus-induced gene silencing to reduce DBOX transcript levels resulted in a corresponding reduction in sanguinarine, dihydrosanguinarine, and papaverine accumulation in opium poppy roots in sup-port of DBOX as a multifunctional oxidative enzyme in BIA metabolism.