Department of Chemistry and Biochemistry, The Astrobiology Biogeocatalysis Research Center, Montana State University, 103 CBB, Bozeman, MT 59717, USA.
J Biol Inorg Chem. 2010 Aug;15(6):943-55. doi: 10.1007/s00775-010-0656-8. Epub 2010 Apr 20.
Spore photoproduct lyase (SP lyase), a member of the radical S-adenosylmethionine superfamily of enzymes, catalyzes the repair of 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)], a type of UV-induced DNA damage unique to bacterial spores. The anaerobic purification and characterization of Clostridium acetobutylicum SP lyase heterologously expressed in Escherichia coli, and its catalytic activity in repairing stereochemically defined synthetic dinucleotide SPs was investigated. The purified enzyme contains between 2.3 and 3.1 iron atoms per protein. Electron paramagnetic resonance (EPR) spectroscopy reveals an isotropic signal centered at g = 1.99, characteristic of a 3Fe-4S cluster accounting for 3-4% of the iron in the sample. Upon reduction, a nearly axial signal (g = 2.03, 1.93 and 1.92) characteristic of a 4Fe-4S cluster is observed that accounts for 34-45% of total iron. Addition of S-adenosylmethionine to the reduced enzyme produces a rhombic signal (g = 2.02, 1.93, 1.82) unique to the S-adenosyl-L: -methionine complex while decreasing the overall EPR intensity. This reduced enzyme is shown to rapidly and completely repair the 5R diastereomer of a synthetic dinucleotide SP with a specific activity of 7.1 +/- 0.6 nmol min(-1) mg(-1), whereas no repair was observed for the 5S diastereomer.
孢子嘧啶光裂合酶 (SP 裂合酶) 是自由基 S-腺苷甲硫氨酸超家族酶的成员，可催化修复 5-胸腺嘧啶-5,6-二氢胸腺嘧啶 [孢子光产物 (SP)]，这是一种独特的细菌孢子中的 UV 诱导的 DNA 损伤。研究了在大肠杆菌中异源表达的梭菌孢子嘧啶光裂合酶的厌氧纯化和特性，以及其修复立体化学定义的合成二核苷酸 SP 的催化活性。纯化的酶每蛋白含有 2.3 到 3.1 个铁原子。电子顺磁共振 (EPR) 光谱显示出一个各向同性信号，中心位于 g = 1.99，这是一个 3Fe-4S 簇的特征，占样品中铁的 3-4%。还原后，观察到一个几乎轴向的信号 (g = 2.03、1.93 和 1.92)，这是一个 4Fe-4S 簇的特征，占总铁的 34-45%。向还原酶中添加 S-腺苷甲硫氨酸会产生一种独特的菱形信号 (g = 2.02、1.93、1.82)，这是 S-腺苷-L：-甲硫氨酸复合物的特征，同时降低整体 EPR 强度。该还原酶被证明能够快速且完全地修复合成二核苷酸 SP 的 5R 非对映异构体，比活为 7.1 +/- 0.6 nmol min(-1) mg(-1)，而对 5S 非对映异构体则没有观察到修复。
