Identification of PpoA from Aspergillus nidulans as a Fusion Protein of a Fatty Acid Heme Dioxygenase/Peroxidase and a Cytochrome P450
Identification of PpoA from Aspergillus nidulans as a Fusion Protein of a Fatty Acid Heme Dioxygenase/Peroxidase and a Cytochrome P450
复制标题
DOI：
10.1074/jbc.m809152200
复制
发表时间：
2009-05-01
影响因子：
4.8
通讯作者：
Feussner, Ivo
中科院分区：
文献类型：
--
作者：
Brodhun, Florian;Goebel, Cornelia;Feussner, Ivo
同宗子囊菌构巢曲霉因其能够以无性和有性生命周期繁殖的能力而成为丝状真菌的模式生物，并且脂肪酸衍生的物质调节两个周期之间的平衡。这些所谓的 psi（性早熟诱导剂）因子是由产生 psi 因子的加氧酶（Ppo 酶）产生的。生物信息分析预测Ppo蛋白中存在两个不同的血红素结构域：在N端区域，预测脂肪酸血红素双加氧酶/过氧化物酶结构域，而在C端区域，预测P450血红素硫醇结构域。为了分析 Ppo 酶催化的反应，PpoA 在大肠杆菌中作为活性酶表达。该蛋白质经过 62 倍纯化，被鉴定为同源四聚铁血红素蛋白，可在 pH 值接近 7.25 的情况下代谢单不饱和 C-16 和 C-18 脂肪酸。根据序列比对和特征性 450 nm CO 结合光谱的出现，证实了硫醇盐连接的血红素的存在。对其反应机制的研究表明，PpoA 使用不同的血红素结构域来催化两个独立的反应。在血红素过氧化物酶结构域内，亚油酸通过从脂肪酸的 C-8 中提取 H 原子而被氧化为 (8R)-氢过氧十八碳二烯酸，产生与分子双氧反应的以碳为中心的自由基。在第二个反应步骤中，8-氢过氧十八碳二烯酸在 P450 血红素硫醇结构域内异构化为 5,8-二羟基十八碳二烯酸。我们将 PpoA 鉴定为一种双功能 P450 融合蛋白，它使用以前未知的反应机制来形成 psi 因子。
The homothallic ascomycete Aspergillus nidulans serves as model organism for filamentous fungi because of its ability to propagate with both asexual and sexual life cycles, and fatty acid-derived substances regulate the balance between both cycles. These so-called psi (precocious sexual inducer) factors are produced by psi factor-producing oxygenases (Ppo enzymes). Bioinformatic analysis predicted the presence of two different heme domains in Ppo proteins: in the N-terminal region, a fatty acid heme dioxygenase/peroxidase domain is predicted, whereas in the C-terminal region, a P450 heme thiolate domain is predicted. To analyze the reaction catalyzed by Ppo enzymes, PpoA was expressed in Escherichia coli as an active enzyme. The protein was purified by 62-fold and identified as a homotetrameric ferric heme protein that metabolizes mono-as well as polyunsaturated C-16 and C-18 fatty acids at pH similar to 7.25. The presence of thiolate-ligated heme was confirmed on the basis of sequence alignments and the appearance of a characteristic 450 nm CO-binding spectrum. Studies on its reaction mechanism revealed that PpoA uses different heme domains to catalyze two separate reactions. Within the heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid by abstracting a H-atom from C-8 of the fatty acid, yielding a carbon-centered radical that reacts with molecular dioxygen. In the second reaction step, 8-hydroperoxyoctadecadienoic acid is isomerized within the P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. We identify PpoA as a bifunctional P450 fusion protein that uses a previously unknown reaction mechanism for forming psi factors.