Bauer Jacob, Ondrovičová Gabriela, Najmanová Lucie, Pevala Vladimír, Kameník Zdeněk, Koštan Július, Janata Jiří, Kutejová Eva
Institute of Molecular Biology, Slovak Academy of Sciences, 851 45 Bratislava, Slovakia.
Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic.
Acta Crystallogr D Biol Crystallogr. 2014 Apr;70(Pt 4):943-57. doi: 10.1107/S139900471303397X. Epub 2014 Mar 19.
The S-adenosyl-L-methionine (SAM)-dependent methyltransferase CcbJ from Streptomyces caelestis catalyzes one of the final steps in the biosynthesis of the antibiotic celesticetin, methylation of the N atom of its proline moiety, which greatly enhances the activity of the antibiotic. Since several celesticetin variants exist, this enzyme may be able to act on a variety of substrates. The structures of CcbJ determined by MAD phasing at 3.0 Å resolution, its native form at 2.7 Å resolution and its complex with S-adenosyl-L-homocysteine (SAH) at 2.9 Å resolution are reported here. Based on these structures, three point mutants, Y9F, Y17F and F117G, were prepared in order to study its behaviour as well as docking simulations of both CcbJ-SAM-substrate and CcbJ-SAH-product complexes. The structures show that CcbJ is a class I SAM-dependent methyltransferase with a wide active site, thereby suggesting that it may accommodate a number of different substrates. The mutation results show that the Y9F and F117G mutants are almost non-functional, while the Y17F mutant has almost half of the wild-type activity. In combination with the docking studies, these results suggest that Tyr9 and Phe117 are likely to help to position the substrate for the methyl-transfer reaction and that Tyr9 may also facilitate the reaction by removing an H(+) ion. Tyr17, on the other hand, seems to operate by helping to stabilize the SAM cofactor.
来自天蓝色链霉菌的S-腺苷-L-甲硫氨酸（SAM）依赖性甲基转移酶CcbJ催化抗生素天蓝色菌素生物合成的最后步骤之一，即其脯氨酸部分的N原子甲基化，这大大增强了抗生素的活性。由于存在几种天蓝色菌素变体，这种酶可能能够作用于多种底物。本文报道了通过MAD相位解析在3.0 Å分辨率下测定的CcbJ结构、在2.7 Å分辨率下测定的其天然形式以及在2.9 Å分辨率下测定的其与S-腺苷-L-高半胱氨酸（SAH）的复合物结构。基于这些结构，制备了三个点突变体Y9F、Y17F和F117G，以研究其行为以及CcbJ-SAM-底物和CcbJ-SAH-产物复合物的对接模拟。结构表明，CcbJ是一种具有宽活性位点的I类SAM依赖性甲基转移酶，因此表明它可能容纳多种不同的底物。突变结果表明，Y9F和F117G突变体几乎无功能，而Y17F突变体具有几乎一半的野生型活性。结合对接研究，这些结果表明Tyr9和Phe117可能有助于将底物定位用于甲基转移反应，并且Tyr9也可能通过去除H(+)离子促进反应。另一方面，Tyr17似乎通过帮助稳定SAM辅因子来发挥作用。
