Plasmid pCAR3 Contains Multiple Gene Sets Involved in the Conversion of Carbazole to Anthranilate
Plasmid pCAR3 Contains Multiple Gene Sets Involved in the Conversion of Carbazole to Anthranilate
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DOI：
10.1128/aem.72.5.3198-3205.2006
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发表时间：
2006-05
影响因子：
4.4
通讯作者：
Masaaki Urata;Hiromasa Uchimura;H. Noguchi;Tomoya Sakaguchi;T. Takemura;Kaori Eto;H. Habe;T. Omori;H. Yamane;H. Nojiri
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作者：
Masaaki Urata;Hiromasa Uchimura;H. Noguchi;Tomoya Sakaguchi;T. Takemura;Kaori Eto;H. Habe;T. Omori;H. Yamane;H. Nojiri
鞘氨单胞菌（Sphingomonas sp.）菌株KA1的咔唑降解car- 1基因簇（carAaIBaIBbICIAcI）位于254 kb的环状质粒pCAR3上。咔唑由咔唑1,9a-双加氧酶（CARDO; CarAaIAcI）、内切酶（caraibbi）和水解酶（CarCI）催化转化为邻氨基苯甲酸酯。CARDO是一种三组分双加氧酶，CarAaI和CarAcI是其末端加氧酶和铁氧还蛋白组分。car- 1基因簇缺乏编码CARDO的铁氧还蛋白还原酶组分的基因。在本研究中，基于pCAR3的草图序列，我们发现了多个咔唑降解基因分散在pCAR3的四个位点上，包括car基因簇的第二个拷贝（carAaIIBaIIBbIICIIAcII）和铁氧还蛋白/还原酶基因fdxI-fdrI和fdrII。生物转化实验表明，FdrI（或FdrII）可以借助铁氧还蛋白（CarAcI、CarAcII或FdxI）驱动NAD(P)H到CarAaI（或CarAaII）的电子转移链。由于这条电子传递链与恶臭假单胞菌P450cam单加氧酶系统中由腐胺还氧蛋白和腐胺还氧蛋白还原酶组成的电子传递链具有亲缘关系，因此KA1的CARDO系统可归为IIA类Rieske非血红素铁加氧酶系统。逆转录- pcr （RT-PCR）和定量RT-PCR分析显示，KA1暴露于咔唑时，两个car基因簇构成了操纵子，尽管fdxI-fdrI和fdrII基因是组成性表达的，但它们的表达受到诱导。KA1的两种末端加氧酶都表现出与咔唑降解物假单胞菌（Pseudomonas resinovorans CA10）大致相同的底物特异性，尽管存在细微差异。
ABSTRACT The carbazole degradative car-I gene cluster (carAaIBaIBbICIAcI) of Sphingomonas sp. strain KA1 is located on the 254-kb circular plasmid pCAR3. Carbazole conversion to anthranilate is catalyzed by carbazole 1,9a-dioxygenase (CARDO; CarAaIAcI), meta-cleavage enzyme (CarBaIBbI), and hydrolase (CarCI). CARDO is a three-component dioxygenase, and CarAaI and CarAcI are its terminal oxygenase and ferredoxin components. The car-I gene cluster lacks the gene encoding the ferredoxin reductase component of CARDO. In the present study, based on the draft sequence of pCAR3, we found multiple carbazole degradation genes dispersed in four loci on pCAR3, including a second copy of the car gene cluster (carAaIIBaIIBbIICIIAcII) and the ferredoxin/reductase genes fdxI-fdrI and fdrII. Biotransformation experiments showed that FdrI (or FdrII) could drive the electron transfer chain from NAD(P)H to CarAaI (or CarAaII) with the aid of ferredoxin (CarAcI, CarAcII, or FdxI). Because this electron transfer chain showed phylogenetic relatedness to that consisting of putidaredoxin and putidaredoxin reductase of the P450cam monooxygenase system of Pseudomonas putida, CARDO systems of KA1 can be classified in the class IIA Rieske non-heme iron oxygenase system. Reverse transcription-PCR (RT-PCR) and quantitative RT-PCR analyses revealed that two car gene clusters constituted operons, and their expression was induced when KA1 was exposed to carbazole, although the fdxI-fdrI and fdrII genes were expressed constitutively. Both terminal oxygenases of KA1 showed roughly the same substrate specificity as that from the well-characterized carbazole degrader Pseudomonas resinovorans CA10, although slight differences were observed.