Identification and Characterization of a Novel-type Ferric Siderophore Reductase from a Gram-positive Extremophile*
Identification and Characterization of a Novel-type Ferric Siderophore Reductase from a Gram-positive Extremophile*
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DOI：
10.1074/jbc.m110.192468
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发表时间：
2010-11
期刊：
影响因子：
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通讯作者：
M. Miethke;A. Pierik;Florian Peuckert;A. Seubert;M. Marahiel
中科院分区：
文献类型：
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作者：
M. Miethke;A. Pierik;Florian Peuckert;A. Seubert;M. Marahiel
铁限制是微生物生命的一个主要限制，大量的微生物使用铁载体进行高亲和力的铁获取。由于尚不清楚革兰氏阳性反应中还原铁释放的特定酶，我们搜索了厚壁菌基因组，发现了一种新的铁铁载体还原酶和摄取基因的关联模式。产生分裂素的嗜碱芽孢杆菌的还原酶被发现与柠檬酸铁-羟酸盐摄取系统聚集在一起，并有效地催化铁从铁[III]-dicitrate，铁[III]-分裂素，铁[III]-有氧肌动蛋白和铁铬铁中释放。因此，该基因被命名为fchR，即柠檬酸铁和羟酸还原酶。FchR的紧密结合的[2Fe-2S]辅因子通过紫外可见光谱、EPR、CD光谱和质谱鉴定。以铁氧还蛋白为电子供体，测定了几种底物对铁的释放动力学。催化效率在铁硫支架蛋白清除释放的亚铁的存在下得到了极大的提高。用Ki值在微摩尔范围内的带Ga（III）电荷的铁载体观察到FchR的竞争性抑制。发现主要的催化机制是底物结合Km和KD值的增加与kcat值的增加相耦合，从而在较宽的氧化还原范围内产生较高的催化效率。生理上，即使在铁载体存在的情况下，染色体fchR缺失也会导致铁限制期间的生长严重受损。ΔfchR的电感耦合血浆质谱分析显示细胞内铁积累，表明铁底物没有有效代谢。我们进一步表明，体内氧化还原惰性铁载体模拟物可以有效地抑制FchR，这表明底物特异性铁载体还原酶可能是微生物病原体控制的未来目标。
Iron limitation is one major constraint of microbial life, and a plethora of microbes use siderophores for high affinity iron acquisition. Because specific enzymes for reductive iron release in Gram-positives are not known, we searched Firmicute genomes and found a novel association pattern of putative ferric siderophore reductases and uptake genes. The reductase from the schizokinen-producing alkaliphile Bacillus halodurans was found to cluster with a ferric citrate-hydroxamate uptake system and to catalyze iron release efficiently from Fe[III]-dicitrate, Fe[III]-schizokinen, Fe[III]-aerobactin, and ferrichrome. The gene was hence named fchR for ferric citrate and hydroxamate reductase. The tightly bound [2Fe-2S] cofactor of FchR was identified by UV-visible, EPR, CD spectroscopy, and mass spectrometry. Iron release kinetics were determined with several substrates by using ferredoxin as electron donor. Catalytic efficiencies were strongly enhanced in the presence of an iron-sulfur scaffold protein scavenging the released ferrous iron. Competitive inhibition of FchR was observed with Ga(III)-charged siderophores with Ki values in the micromolar range. The principal catalytic mechanism was found to couple increasing Km and KD values of substrate binding with increasing kcat values, resulting in high catalytic efficiencies over a wide redox range. Physiologically, a chromosomal fchR deletion led to strongly impaired growth during iron limitation even in the presence of ferric siderophores. Inductively coupled plasma-MS analysis of ΔfchR revealed intracellular iron accumulation, indicating that the ferric substrates were not efficiently metabolized. We further show that FchR can be efficiently inhibited by redox-inert siderophore mimics in vivo, suggesting that substrate-specific ferric siderophore reductases may present future targets for microbial pathogen control.