Unveiling the genes responsible for the unique Pseudomonas aeruginosa oleate-diol synthase activity
Unveiling the genes responsible for the unique Pseudomonas aeruginosa oleate-diol synthase activity
复制标题
DOI：
10.1016/j.bbalip.2014.06.010
复制
发表时间：
2014-10-01
影响因子：
4.8
通讯作者：
Manresa, Angels
中科院分区：
文献类型：
--
作者：
Estupinan, Monica;Diaz, Pilar;Manresa, Angels
铜绿假单胞菌显示出将油酸生物转化为一类称为氧脂的羟基化脂肪酸的能力。二醇合酶活性负责这种转化，其通过油酸的二氧合以释放氢过氧化物10-H（P）OME（（10 S）-羟基-（8 E）-十八碳烯酸），随后将氢过氧化物中间体转化为7，10-DiHOME（（75，10 S）-二羟基-（8 E）-十八碳烯酸）进行，这两者都在培养物上清液中积累。分析了铜绿假单胞菌PAW的几种突变体的10-H（P）OME和7，10-DiHOME的产生，检测到其中两种（ORF PA 2077和PA 2078）不能释放羟基化脂肪酸，并选择其用于进一步分析。ORFs PA 2077和PA 2078参与油酸-二醇合成酶的活性得到证实，并通过突变互补分析了它们各自在油酸转化中的作用。活性恢复显示基因PA 2077编码负责反应第一步的10 S-双加氧酶活性（10 S-DOX），而PA 2078编码允许10-H（P）OME转化为7，10-DiHOME的（7S，10 S）-氢过氧化物二醇合酶（7，10-DS）。这两种酶的异源表达分别表明，没有异源复合物的形成所需的酶活性。生物信息学和RT-PCR分析表明，这两个基因构成了新的精细调节油酸二醇合酶操纵子，起源于基因复制事件，然后通过新功能化来适应环境，这在原核生物中是前所未有的。(C)2014爱思唯尔有限公司版权所有。
Pseudomonas aeruginosa displays the ability to perform bioconversion of oleic acid into a class of hydroxylated fatty acids known as oxylipins. A diol synthase activity is responsible for such a conversion, which proceeds through the dioxygenation of oleic acid to release hydroperoxide 10-H(P)OME ((10S)-hydroxy-(8E)-octadecenoic acid), followed by conversion of the hydroperoxide intermediate into 7,10-DiHOME ((75,10S)-dihydroxy-(8E)-octadecenoic acid), both of which accumulate in the culture supernatant. Several mutants of P. aeruginosa PAW were analyzed for the production of 10-H(P)OME and 7,10-DiHOME and two of them (ORFs PA2077 and PA2078), unable to release hydroxylated fatty adds, were detected and selected for further analysis. Involvement of ORFs PA2077 and PA2078 in oleate-diol synthase activity was confirmed, and their respective role in the conversion of oleic acid was analyzed by mutation complementation. Activity restoration revealed that gene PA2077 codes for the 10S-dioxygenase activity (10S-DOX) responsible for the first step of the reaction, whereas PA2078 encodes for the (7S,10S)-hydroperoxide diol synthase enzyme (7,10-DS) which allows the conversion of 10-H(P)OME into 7,10-DiHOME. Heterologous expression of both enzymes separately showed that no hetero-complex formation is required for enzymatic activity. Bioinformatics and RT-PCR analysis revealed that both genes constitute anew fine regulated oleate-diol synthase operon, originated by a gene duplication event followed by neofunctionalization for environmental adaptation, being unprecedented in prokaryotes. (C) 2014 Elsevier B.V. All rights reserved.