Substrate Specificity of Purified Recombinant Chicken -Carotene 9,10-Oxygenase (BCO2)
Substrate Specificity of Purified Recombinant Chicken -Carotene 9,10-Oxygenase (BCO2)
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DOI：
10.1074/jbc.m116.723684
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发表时间：
2016-07-08
影响因子：
4.8
通讯作者：
Harrison, Earl H.
中科院分区：
文献类型：
--
作者：
dela Sena, Carlo;Sun, Jian;Harrison, Earl H.
维生素A原类胡萝卜素被-胡萝卜素15,15-双加氧酶（BCO1）在中心的15-15双键处氧化裂解，形成视网膜（维生素A醛）。另一种类胡萝卜素加氧酶-胡萝卜素9,10-加氧酶（BCO2）催化类胡萝卜素在9-10键处的氧化裂解，生成一个离子酮和一个apoo -10-类胡萝卜素。先前发表的BCO2底物特异性研究是使用表达重组BCO2的细菌或昆虫细胞的粗裂解物进行的。我们试图获得在大肠杆菌中表达的活性重组人BCO2，但没有成功。我们在大肠杆菌BL21-Gold （DE3）中表达了重组鸡BCO2，并通过钴离子亲和层析纯化了该酶。和BCO1一样，纯化的重组鸡BCO2可以催化维生素A原类胡萝卜素-胡萝卜素、-胡萝卜素和-隐黄质的氧化裂解。其以-胡萝卜素为底物的催化活性比BCO1至少低10倍。与BCO1相比，纯化的重组鸡BCO2还能催化9-顺式胡萝卜素、非维生素A原类胡萝卜素玉米黄质和叶黄素的氧化裂解，对全反式番茄红素和类伪胡萝卜素无活性。HPLC法检测到酶促产物为类载脂蛋白10-胡萝卜素，LC-MS法鉴定。在BCO2-隐黄质反应混合物中也持续检测到少量的3-羟基-载8-胡萝卜素。除了与-隐黄素的这种活性外，BCO2专门在9-10键上裂解产生-10-类胡萝卜素。BCO2已被证明具有防止类胡萝卜素过度积累的功能，其广泛的底物特异性与此一致。
Provitamin A carotenoids are oxidatively cleaved by -carotene 15,15-dioxygenase (BCO1) at the central 15-15 double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, -carotene 9,10-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9-10 bond to yield an ionone and an apo-10-carotenoid. Previously published substrate specificity studies of BCO2 were conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in Escherichia coli were unsuccessful. We have expressed recombinant chicken BCO2 in the strain E. coli BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids -carotene, -carotene, and -cryptoxanthin. Its catalytic activity with -carotene as substrate is at least 10-fold lower than that of BCO1. In further contrast to BCO1, purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis--carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and -apocarotenoids. Apo-10-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3-hydroxy--apo-8-carotenal were also consistently detected in BCO2--cryptoxanthin reaction mixtures. With the exception of this activity with -cryptoxanthin, BCO2 cleaves specifically at the 9-10 bond to produce apo-10-carotenoids. BCO2 has been shown to function in preventing the excessive accumulation of carotenoids, and its broad substrate specificity is consistent with this.