Characterization of a new thermophilic spore photoproduct lyase from geobacillus stearothermophilus (SplG) with defined lesion containing DNA substrates
Characterization of a new thermophilic spore photoproduct lyase from geobacillus stearothermophilus (SplG) with defined lesion containing DNA substrates
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DOI：
10.1074/jbc.m607053200
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发表时间：
2006-11-24
影响因子：
4.8
通讯作者：
Carell, Thomas
中科院分区：
文献类型：
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作者：
Pieck, J. Carsten;Hennecke, Ulrich;Carell, Thomas
嗜热脂肪地杆菌SplG基因编码一种嗜热孢子光产物裂解酶(SplG)，属于自由基酶S-腺苷甲硫氨酸(ADOMet)家族。在有氧条件下纯化的apo-SplG形成同源二聚体，每个单体单位在重组为全貌后含有一个[4Fe-4S]簇。通过对每个均二聚体的铁和硫量的定量以及UV和EPR光谱的测定，证明了[4Fe-4S]团簇的形成。紫外光谱特征是典型的[4Fe-4S]团簇在420 nm处的特征吸收，EPR数据与其他含有[4Fe-4S](+)中心的蛋白质的EPR数据相同。用含有一个孢子光产物损伤的寡核苷酸检测全息SplG的活性，证明了该酶能够翻转底物。除了修复外，我们还观察到了ADOMet的裂解生成5‘-脱氧腺苷。在Aza-ADOMet存在下，SplG被完全抑制，这为修复机制提供了直接支持。
The Geobacillus stearothermophilus splG gene encodes a thermophilic spore photoproduct lyase (SplG) that belongs to the family of radical S-adenosylmethionine ( AdoMet) enzymes. The aerobically purified apo-SplG forms a homodimer, which contains one [4Fe-4S] cluster per monomer unit after reconstitution to the holoform. Formation of the [4Fe-4S] cluster was proven by quantification of the amount of iron and sulfur per homodimer and by UV and EPR spectroscopy. The UV spectrum features a characteristic absorbance at 420 nm typical for [4Fe-4S] clusters, and the EPR data were found to be identical to those of other proteins containing an[4Fe-4S](+) center. Probing of the activity of the holo-SplG with oligonucleotides containing one spore photoproduct lesion at a defined site proved that the enzyme is able to turn over substrate. In addition to repair, we observed cleavage of AdoMet to generate 5'-deoxyadenosine. In the presence of aza-AdoMet the SplG is completely inhibited, which provides direct support for the repair mechanism.