Institute of Industrial Genetics, University of Stuttgart, Stuttgart, Germany.
FEBS J. 2012 Oct;279(20):3911-24. doi: 10.1111/j.1742-4658.2012.08751.x. Epub 2012 Sep 11.
Carotenoid cleavage oxygenases are nonheme iron enzymes that specifically cleave carbon-carbon double bonds of carotenoids. Their apocarotenoid cleavage products serve as important signaling molecules that are involved in various biological processes. A database search revealed the presence of putative carotenoid cleavage oxygenase genes in the genomes of Sphingopyxis alaskensis RB2256 and Plesiocystis pacifica SIR-1. The four genes sala_1698, sala_1008, ppsir1_15490 and ppsir1_17230 were cloned and heterologously expressed in carotenoid-producing Escherichia coli JM109 strains. Two of the four encoded proteins exhibited carotenoid cleavage activity. S. alaskensis RB2256 carotenoid cleavage oxygenase (SaCCO), which is encoded by sala_1698, was shown to cleave acyclic and monocyclic substrates. Coexpression of sala_1698 in carotenoid-producing E. coli JM109 strains revealed cleavage activity for lycopene, hydroxylycopene, and dihydroxylycopene. The monocyclic substrate apo-8'-carotenal was cleaved in vitro by purified SaCCO at the 9'/10' and 11'/12' double bonds. The second enzyme, P. pacifica SIR-1 carotenoid cleavage oxygenase (PpCCO), is encoded by ppsir1_15490. PpCCO-mediated carotenoid cleavage requires the presence of either hydroxy or keto groups. PpCCO cleaved zeaxanthin, hydroxylycopene, and dihydroxylycopene, and also the C(50) carotenoids decaprenoxanthin, sarprenoxanthin and sarcinaxanthin, in carotenoid-producing E. coli JM109 strains. Whole cells of E. coli JM109 overexpressing ppsir1_15490mut, a mutant of ppsir1_15490 with enhanced gene expression, were applied for the conversion of carotenoids. Analysis of the carotenoid cleavage products revealed a single cleavage site at the 13'/14' double bond for astaxanthin, and two cleavage sites at the 11'/12' or 13'/14' double bond for zeaxanthin, nostoxanthin, and canthaxanthin.
类胡萝卜素裂解加氧酶是一类非血红素铁酶，能够特异性地裂解类胡萝卜素中的碳-碳双键。它们的副产物可作为重要的信号分子，参与各种生物过程。数据库搜索显示，在鞘氨醇单胞菌 RB2256 和太平洋小球藻 SIR-1 的基因组中存在假定的类胡萝卜素裂解加氧酶基因。克隆了 sala_1698、sala_1008、ppsir1_15490 和 ppsir1_17230 这四个基因，并在产类胡萝卜素的大肠杆菌 JM109 菌株中进行了异源表达。这四个编码蛋白中的两个表现出类胡萝卜素裂解活性。由 sala_1698 编码的鞘氨醇单胞菌 RB2256 类胡萝卜素裂解加氧酶（SaCCO）被证明能够裂解无环和单环底物。在产类胡萝卜素的大肠杆菌 JM109 菌株中共同表达 sala_1698 可使番茄红素、羟基番茄红素和二羟基番茄红素发生裂解。体外纯化的 SaCCO 可裂解单环底物 apo-8'-胡萝卜醛在 9'/10'和 11'/12'双键处。第二种酶，太平洋小球藻 SIR-1 类胡萝卜素裂解加氧酶（PpCCO），由 ppsir1_15490 编码。PpCCO 介导的类胡萝卜素裂解需要羟基或酮基的存在。PpCCO 可裂解玉米黄质、羟基番茄红素和二羟基番茄红素，以及产类胡萝卜素的大肠杆菌 JM109 菌株中的 C(50)类胡萝卜素脱甲藻叶黄素、鲨烯叶黄素和岩藻叶黄素。过表达 ppsir1_15490mut 的大肠杆菌 JM109 全细胞突变株，其为 ppsir1_15490 的一个增强基因表达的突变体，可用于类胡萝卜素的转化。对类胡萝卜素裂解产物的分析表明，虾青素在 13'/14'双键处只有一个裂解位点，而玉米黄质、角黄素和叶黄素在 11'/12'或 13'/14'双键处有两个裂解位点。
