Regulation of the NADP-glutamate dehydrogenase gene gdhA in Aspergillus nidulans by the Zn(II)2Cys6 transcription factor LeuB
Regulation of the NADP-glutamate dehydrogenase gene gdhA in Aspergillus nidulans by the Zn(II)2Cys6 transcription factor LeuB
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DOI：
10.1099/mic.0.071514-0
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发表时间：
2013-12-01
期刊：
影响因子：
2.8
通讯作者：
Todd, Richard B.
中科院分区：
文献类型：
--
作者：
Downes, Damien J.;Dayis, Meryl A.;Todd, Richard B.
NADP依赖的谷氨酸脱氢酶（NADP-GDH）是真菌通过铵同化替代氮营养源的关键酶。在构巢曲霉中，NADP-GDH由gdhA编码。已知几种转录因子调节gdhA表达，包括氮代谢基因的主要转录激活因子AreA和AreA的共激活因子TamA。TamA还与亮氨酸生物合成的调节剂LeuB相互作用。我们研究了亮氨酸生物合成对gdhA调节的影响，发现亮氨酸调节NADP-GDH活性和gdhA表达水平。我们表明，使用突变体与扰动水平的α-异丙基苹果酸（α-IPM），这亮氨酸生物合成中间体影响gdhA调节。亮氨酸调节gdhA需要一个功能性LeuB与完整的Zn（II）2Cys 6 DNA结合域。通过分析gdhA直系同源物启动子中假定LeuB DNA结合位点的流行情况，我们预测除了镰刀菌和裂殖酵母外，子囊菌内NADP-GDH表达的亮氨酸调节广泛保守。利用gdhA-lacZ报告基因的启动子突变，我们鉴定了LeuB在A.构巢菌gdhA启动子。这两个位点缺乏序列同一性，其中一个位点符合预测的LeuB DNA结合位点共有基序，而第二个位点是曲霉gdhA启动子中保守的新型调控序列元件。这些数据表明，LeuB调节NADP-GDH表达响应亮氨酸水平，这可能是一个重要的传感器的氮可用性。
NADP-dependent glutamate dehydrogenase (NADP-GDH) is a key enzyme in the assimilation of alternative nitrogen nutrient sources through ammonium in fungi. In Aspergillus nidulans, NADP-GDH is encoded by gdhA. Several transcription factors are known to regulate gdhA expression, including AreA, the major transcription activator of nitrogen metabolic genes, and TamA, a co-activator of AreA. TamA also interacts with LeuB, the regulator of leucine biosynthesis. We have investigated the effects of leucine biosynthesis on gdhA regulation, and found that leucine regulates the levels of NADP-GDH activity and gdhA expression. We show, using mutants with perturbed levels of a-isopropylmalate (alpha-IPM), that this leucine biosynthesis intermediate affects gdhA regulation. Leucine regulation of gdhA requires a functional LeuB with an intact Zn(II)2Cys6 DNA-binding domain. By analysing the prevalence of putative LeuB DNA-binding sites in promoters of gdhA orthologues we predict broad conservation of leucine regulation of NADP-GDH expression within ascomycetes except in the fusaria and fission yeasts. Using promoter mutations in gdhA-lacZ reporter genes we identified two sites of action for LeuB within the A. nidulans gdhA promoter. These two sites lack sequence identity, with one site conforming to the predicted LeuB DNA-binding site consensus motif, whereas the second site is a novel regulatory sequence element conserved in Aspergillus gdhA promoters. These data suggest that LeuB regulates NADP-GDH expression in response to leucine levels, which may act as an important sensor of nitrogen availability.