Identification of dioxygenases required for aspergillus development -: Studies of products, stereochemistry, and the reaction mechanism
Identification of dioxygenases required for aspergillus development -: Studies of products, stereochemistry, and the reaction mechanism
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DOI：
10.1074/jbc.m705366200
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发表时间：
2007-11-30
影响因子：
4.8
通讯作者：
Oliw, Ernst H.
中科院分区：
文献类型：
--
作者：
Garscha, Ulrike;Jerneren, Fredrik;Oliw, Ernst H.
曲霉属含有ppoA、ppoB和ppoC基因，其编码与真菌亚油酸7，8-二醇脱氢酶（7，8-LDS）和环加氧酶具有同源性的脂肪酸加氧酶。我们的目标是确定这些酶，因为PPO基因替换显示出人类病原体烟曲霉和遗传模型构巢曲霉中孢子形成和致病性的关键发育异常。A.烟曲霉和A. nidulans被鉴定为具有氢过氧化物异构酶活性的（8 R）-双加氧酶，命名为5，8-LDS。5，8-LDS将18：2n-6转化为（8 R）-氢过氧十八碳二烯酸（（8 R）- HPODE）和（5S，8 R）-二羟基-9Z，12 Z-十八碳二烯酸（（5S，8 R）-DiHODE）。我们还在A中检测到8，11-LDS。烟曲霉和（10 R）-双加氧酶。二醇脱氢酶将[（8 R）-2H] 18：2n-6氧化为（8 R）-HPODE，并保留氘标记，表明反面氢提取和分子氧插入。用立体特异性氘代18：2n-6进行的实验表明，（8 R）HPODE通过5，8-和8，11-LDS分别异构化为（5S，8 R）DiHODE和（8 R，11 S）-二羟基-9Z，12 Z-十八碳二烯酸，通过在C-5和C-11的超表面氢提取和氧插入。PpoCs被鉴定为（10 R）-双加氧酶，其催化18：2n-6的C-8处的pro-S氢的提取、双键迁移和分子氧的对面插入，形成（10 R）-羟基8 E，12 Z-氢过氧十八碳二烯酸（（10 R）-HPODE）。ppoA的缺失导致（8 R）- H（P）ODE的显著减少和（5S，8 R）-DiHODE生物合成的完全丧失，而（10 R）-HPODE的生物合成不受影响。ppoC的缺失导致痕量外消旋10-HODE的生物合成，但不影响其他氧化脂质的生物合成。我们的结论是，曲霉属的ppoA可能编码5，8-LDS，与7，8-LDS和亚油酸（10 R）-双加氧酶的ppoC具有催化相似性。这些加氧酶及其产物的鉴定将提供工具，用于分析的生物学影响的氧脂素的生物合成中的牛肝菌。
Aspergillus sp. contain ppoA, ppoB, and ppoC genes, which code for fatty acid oxygenases with homology to fungal linoleate 7,8-diol synthases (7,8-LDS) and cyclooxygenases. Our objective was to identify these enzymes, as ppo gene replacements show critical developmental aberrancies in sporulation and pathogenicity in the human pathogen Aspergillus fumigatus and the genetic model Aspergillus nidulans. The PpoAs of A. fumigatus and A. nidulans were identified as (8R)-dioxygenases with hydroperoxide isomerase activity, designated 5,8-LDS. 5,8-LDS transformed 18:2n-6 to (8R)-hydroperoxyoctadecadienoic acid ((8R)- HPODE) and (5S,8R)-dihydroxy-9Z,12Z-octadecadienoic acid ((5S,8R)-DiHODE). We also detected 8,11-LDS in A. fumigatus and (10R)-dioxygenases in both Aspergilli. The diol synthases oxidized [(8R)-2H] 18: 2n-6 to (8R)-HPODE with retention of the deuterium label, suggesting antarafacial hydrogen abstraction and insertion of molecular oxygen. Experiments with stereospecifically deuterated 18: 2n-6 showed that (8R)HPODE was isomerized by 5,8- and 8,11-LDS to (5S, 8R)DiHODE and to (8R,11S)-dihydroxy-9Z, 12Z-octadecadienoic acid, respectively, by suprafacial hydrogen abstraction and oxygen insertion at C-5 and C-11. PpoCs were identified as (10R)-dioxygenases, which catalyzed abstraction of the pro-S hydrogen at C-8 of 18: 2n-6, double bond migration, and antafacial insertion of molecular oxygen with formation of (10R)-hydroxy8E, 12Z-hydroperoxyoctadecadienoic acid ((10R)-HPODE). Deletion of ppoA led to prominent reduction of (8R)- H(P) ODE and complete loss of (5S,8R)-DiHODE biosynthesis, whereas biosynthesis of (10R)-HPODE was unaffected. Deletion of ppoC caused biosynthesis of traces of racemic 10-HODE but did not affect the biosynthesis of other oxylipins. We conclude that ppoA of Aspergillus sp. may code for 5,8-LDS with catalytic similarities to 7,8-LDS and ppoC for linoleate (10R)-dioxygenases. Identification of these oxygenases and their products will provide tools for analyzing the biological impact of oxylipin biosynthesis in Aspergilli.