Organization and function of the YsiA regulon of Bacillus subtilis involved in fatty acid degradation
Organization and function of the YsiA regulon of Bacillus subtilis involved in fatty acid degradation
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DOI：
10.1074/jbc.m606831200
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发表时间：
2007-02-23
影响因子：
4.8
通讯作者：
Fujita, Yasutaro
中科院分区：
文献类型：
--
作者：
Matsuoka, Hiroshi;Hirooka, Kazutake;Fujita, Yasutaro
研究了枯草芽孢杆菌YsiA调节子在脂肪酸降解中的组织和功能。北方和引物延伸分析表明，该调节子包含5个操纵子，即lcfA-ysiA-B-etfB-A、ykuF-G、yhfL、yusM-L-K-J和ywjF-acdA-rpoE。推测YusJ和AcdA、YsiB和YusL以及YusK分别编码酰基-CoA脱氢酶、3-羟基-CoA脱氢酶/烯酰-CoA水合酶复合物和乙酰-CoA C-酰基转移酶，其直接参与脂肪酸β-氧化循环。此外，LcfA和YhfL可能编码长链酰基辅酶A连接酶。在涉及纯化的YsiA蛋白的凝胶阻滞和足迹分析中，我们确定了YsiA结合的顺式序列（YsiA盒）在ysiA，ykuF，yusL，yhfL和ywjF的启动子区上游，YsiA结合的平衡解离常数（Kd）分别为20，21，37，43和65 nm。YsiA结合被具有14-20个碳原子的长链酰基-CoA特异性抑制，具有18个碳原子的酰基-CoA更有效;在测试的长链酰基-CoA中，单不饱和油酰基-CoA和支链12-甲基十四酰基-CoA最有效。这些体外发现得到了体内观察结果的支持，即通过yusJ、et A或etfB破坏敲除酰基辅酶A脱氢导致YsiA失活，这可能是因为细胞中长链酰基辅酶A的积累。此外，yusL、yusK、yusJ、etfA、etfB或ykuG的破坏影响了棕榈酸（一种代表性的长链脂肪酸）的利用。基于这项工作，ysiA、ysiB、.ykuF、ykuG、yhjL、yusM、yusL、yusK、yusJ和ywjF可以重命名为fadR、fadB、fadH、fadG、lcfB、fadM、fadN、fadA、fadE和fadF。
The organization and function of the Bacillus subtilis YsiA regulon involved in fatty acid degradation were investigated. Northern and primer extension analyses indicated that this regulon comprises five operons, i.e. lcfA-ysiA-B-etfB-A, ykuF-G, yhfL, yusM-L-K-J, and ywjF-acdA-rpoE. YusJ and AcdA, YsiB and YusL, and YusK presumably encode acyl-CoA dehydrogenases, 3-hydroxyl-CoA dehydrogenase/enoyl-CoA hydratase complexes, and acetyl-CoA C-acyltransferase, respectively, which are directly involved in the fatty acid,beta-oxidation cycle. In addition, LcfA and YhfL are likely to encode long chain acyl-CoA ligases. On gel retardation and footprinting analyses involving the purified YsiA protein, we identified cis-sequences for YsiA binding (YsiA boxes) in the promoter regions upstream of ysiA, ykuF, yusL, yhfL, and ywjF, the equilibrium dissociation constants (K-d) for YsiA binding being 20, 21, 37, 43, and 65 nm, respectively. YsiA binding was specifically inhibited by long chain acyl-CoAs with 14-20 carbon atoms, acyl-CoAs with 18 carbon atoms being more effective; out of long chain acyl-CoAs tested, monounsaturated oleoyl-CoA, and branched chain 12-metyltetradecanoyl-CoA were most effective. These in vitro findings were supported by the in vivo observation that the knock-out of acyl-CoA dehydrogenation through yusJ, et A, or etfB disruption resulted in YsiA inactivation, probably because of the accumulation of long chain acyl-CoAs in the cells. Furthermore, the disruption of yusL, yusK, yusJ, etfA, etfB, or ykuG affected the utilization of palmitic acid, a representative long chain fatty acid. Based on this work, ysiA, ysiB,.ykuF, ykuG, yhjL, yusM, yusL, yusK, yusJ, and ywjF can be renamed fadR, fadB, fadH,fadG, lcfB, fadM, fadN, fadA, fadE, and fadF.