Involvement of the nadA gene in formation of G-group aflatoxins in Aspergillus parasiticus
Involvement of the nadA gene in formation of G-group aflatoxins in Aspergillus parasiticus
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DOI：
10.1016/j.fgb.2008.03.003
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发表时间：
2008-07-01
影响因子：
3
通讯作者：
Yabe, Kimiko
中科院分区：
文献类型：
--
作者：
Cai, Jingjing;Zeng, Hongmei;Yabe, Kimiko
nadA基因存在于寄生曲霉和黄曲霉基因组中黄曲霉毒素基因簇的末端。RT-PCR分析表明，nadA基因在黄曲霉毒素诱导的YES培养基中表达，而在黄曲霉毒素非诱导的YEP培养基中不表达。nadA基因在aflR基因缺失突变体中不表达，与所使用的培养基无关。为了阐明nadA基因的功能，我们在黄曲霉毒素A.寄生虫所分离的四个nadA缺失突变体通常在菌丝体和培养基中积累一种新的黄色荧光色素（命名为NADA）。当突变株和野生型菌株在YES培养基中培养3天时，突变株各自产生的G-组黄曲霉毒素的量为野生型菌株产生的量的约50%。相反，B组黄曲霉毒素的量在突变体和野生型菌株之间没有显著差异。NADA色素不稳定，可非酶促转化为黄曲霉毒素G（1）（AFG（1））。LC-MS测定表明NADA的相对分子质量为360，比AFG（1）的相对分子质量高32。我们以前报道过，在无细胞系统中，O-甲基杂色曲霉素（OMST）形成AFG（1）至少需要一种胞浆酶和另外两种微粒体酶。寄生虫本研究证实，野生型A。寄生虫菌株显著促进了OMST中AFG（1）的形成，而nadA的胞质组分则显著促进了OMST中AFG（1）的形成。缺失突变体没有表现出相同的活性。此外，野生型菌株的胞质组分显示催化从NADA到AFG（1）的反应的酶活性，该反应需要NADPH或NADH，表明NADA是AFG（1）的前体;相反，nadA缺失突变体的胞质组分没有显示相同的酶活性。这些结果表明，NadA蛋白是从OMST生物合成G-黄曲霉毒素所需的胞质酶，并且它催化从NADA到AFG的反应（1），即G-黄曲霉毒素生物合成的最后一步。(C)2008年爱思唯尔公司All rights reserved.
The nadA gene is present at the end of the aflatoxin gene cluster in the genome of Aspergillus parasiticus as well as in Aspergillus flavus. RT-PCR analyses showed that the nadA gene was expressed in an aflatoxin-inducible YES medium, but not in an aflatoxin-non-inducible YEP medium. The nadA gene was not expressed in the aflR gene-deletion mutant, irrespective of the culture medium used. To clarify the nadA gene's function, we disrupted the gene in aflatoxigenic A. parasiticus. The four nadA-deletion mutants that were isolated commonly accumulated a novel yellow-fluorescent pigment (named NADA) in mycelia as well as in culture medium. When the mutants and the wild-type strain were cultured for 3 days in YES medium, the mutants each produced about 50% of the amounts of G-group aflatoxins that the wild-type strain produced. In contrast, the amounts of B-group aflatoxins did not significantly differ between the mutants and the wild-type strain. The NADA pigment was so unstable that it could non-enzymatically change to aflatoxin G(1) (AFG(1)). LC-MS measurement showed that the molecular mass of NADA was 360, which is 32 higher than that of AFG(1). We previously reported that at least one cytosol enzyme, together with two other microsome enzymes, is necessary for the formation of AFG(1) from O-methylsterigmatocystin (OMST) in the cell-free system of A. parasiticus. The present study confirmed that the cytosol fraction of the wild-type A. parasiticus strain significantly enhanced the AFG(1) formation from OMST, whereas the cytosol fraction of the nadA. deletion mutant did not show the same activity. Furthermore, the cytosol fraction of the wild-type strain showed the enzyme activity catalyzing the reaction from NADA to AFG(1), which required NADPH or NADH, indicating that NADA is a precursor of AFG(1); in contrast, the cytosol fraction of the nadA-deletion mutant did not show the same enzyme activity. These results demonstrated that the NadA protein is the cytosol enzyme required for G-aflatoxin biosynthesis from OMST, and that it catalyzes the reaction from NADA to AFG(1), the last step in G-aflatoxin biosynthesis. (C) 2008 Elsevier Inc. All rights reserved.