Yamada Osamu, Na Nan Suthamas, Akao Takeshi, Tominaga Mihoko, Watanabe Hisayuki, Satoh Toshitsugu, Enei Hitoshi, Akita Osamu
National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima 739-0046, Japan.
J Biosci Bioeng. 2003;95(1):82-8. doi: 10.1016/S1389-1723(03)80153-6.
We identified and analyzed the dffA gene from Aspergillus oryzae which encodes L-ornithine N5-oxygenase involved in the biosynthesis of deferriferrichrysin, a type of siderophore which is a low-molecular-weight iron chelating compound. From among more than 20,000 clones in an A. oryzae EST (expressed sequence tag) library, we found only one clone encoding a protein that exhibited homology to theUstilago maydis sid1 protein (Sid1) and Pseudomonas aeruginosa pvdA protein (PvdA), both known as the only examples of L-ornithine N5-oxygenase. The complete gene sequence shows that the dffA gene includes a 1575-bp open reading frame (ORF), one 66-bp intorn, which is a typical intorn length inA. oryzae, and encodes 502 amino acids with putative FAD-binding, NADP-binding, and 'FATGY' motifs, which are conserved inN-hydroxylating enzymes. As well as that of the U. maydis sid1 gene,dffA gene expression was induced under iron-limited conditions, and the promoter region has several GATA-type transcription regulator binding motifs. When the dffA gene was expressed under the control of the a-amylase promoter in A. oryzae, transformants revealed inducible high L-ornithine N5-oxygenase activities. In addition, a dffA gene disruptant showed no deferriferrichrysin production even under iron-limited conditions. These results clearly suggest that the dffA gene is indispensable for deferriferrichrysin biosynthesis in A. oryzae.
我们从米曲霉中鉴定并分析了dffA基因，该基因编码参与脱铁富里西林生物合成的L-鸟氨酸N5-加氧酶，脱铁富里西林是一种铁载体，属于低分子量铁螯合化合物。在米曲霉EST（表达序列标签）文库的20000多个克隆中，我们仅发现一个克隆编码的蛋白质与玉米黑粉菌的Sid1蛋白和铜绿假单胞菌的PvdA蛋白具有同源性，这两种蛋白均是已知的L-鸟氨酸N5-加氧酶的唯一实例。完整的基因序列表明，dffA基因包含一个1575 bp的开放阅读框（ORF）、一个66 bp的内含子（这是米曲霉中典型的内含子长度），编码502个氨基酸，具有推定的FAD结合、NADP结合和“FATGY”基序，这些基序在N-羟基化酶中是保守的。与玉米黑粉菌的sid1基因一样，dffA基因的表达在铁限制条件下被诱导，并且启动子区域有几个GATA型转录调节因子结合基序。当dffA基因在米曲霉的α-淀粉酶启动子控制下表达时，转化体显示出可诱导的高L-鸟氨酸N5-加氧酶活性。此外，dffA基因破坏株即使在铁限制条件下也不产生脱铁富里西林。这些结果清楚地表明，dffA基因对于米曲霉中脱铁富里西林的生物合成是必不可少的。
