Spectroscopic characterization and mechanistic investigation of P-methyl transfer by a radical SAM enzyme from the marine bacterium Shewanella denitrificans OS217.
Spectroscopic characterization and mechanistic investigation of P-methyl transfer by a radical SAM enzyme from the marine bacterium Shewanella denitrificans OS217.
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DOI：
10.1016/j.bbapap.2014.09.009
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发表时间：
2014-12
影响因子：
3.2
通讯作者：
Wang, Susan C.
中科院分区：
文献类型：
--
作者：
Allen, Kylie D.;Wang, Susan C.
含有碳-磷键的天然产物在许多生物体中具有重要的生物活性。L-膦丝菌素含有唯一已知的天然存在的碳-磷-碳键连接。在放线菌中，钴胺素依赖性自由基S-腺苷-L-甲硫氨酸（SAM）甲基转移酶PhpK催化第二个C-P键的形成，以产生膦丝菌素中完整的C-P-C键。在这里，我们使用电子顺磁共振和核磁共振光谱表征和证明钴胺素依赖的自由基SAM甲基转移酶表示SD_1168从希瓦氏菌OS 217，海洋细菌，尚未报道合成膦丝菌素的活性。重组、重折叠和重构的SD_1168结合与SAM和钴胺素相互作用的四铁四硫簇。在SAM、还原剂和甲钴胺的存在下，SD_1168令人惊讶地催化N-乙酰基-去甲基膦丝菌素和去甲基膦丝菌素的P-甲基化以分别产生N-乙酰基-膦丝菌素和膦丝菌素。此外，如果提供强还原剂柠檬酸钛（III）和羟钴胺素，则该酶在不存在甲钴胺素的情况下具有活性。当与[甲基-13 C]钴胺素和柠檬酸钛孵育时，产生[甲基-13 C]和未标记的N-乙酰膦丝菌素。我们的研究结果表明，SD_1168催化P-甲基化使用自由基SAM依赖性化学与钴胺素作为辅酶。根据最近的基因组信息，这种P-甲基转移酶的发现表明，S。Escherichia coli产生一种次膦酸盐天然产物。
Natural products containing carbon-phosphorus bonds elicit important bioactivity in many organisms. L-phosphinothricin contains the only known naturally-occurring carbon-phosphorus-carbon bond linkage. In actinomycetes, the cobalamin-dependent radical S-adenosyl-L-methionine (SAM) methyltransferase PhpK catalyzes the formation of the second C-P bond to generate the complete C-P-C linkage in phosphinothricin. Here we use electron paramagnetic resonance and nuclear magnetic resonance spectroscopies to characterize and demonstrate the activity of a cobalamin-dependent radical SAM methyltransferase denoted SD_1168 from Shewanella denitrificans OS217, a marine bacterium that has not been reported to synthesize phosphinothricin. Recombinant, refolded, and reconstituted SD_1168 binds a four-iron, four-sulfur cluster that interacts with SAM and cobalamin. In the presence of SAM, a reductant, and methylcobalamin, SD_1168 surprisingly catalyzes the P-methylation of N-acetyl-demethylphosphinothricin and demethylphosphinothricin to produce N-acetyl-phosphinothricin and phosphinothricin, respectively. In addition, this enzyme is active in the absence of methylcobalamin if the strong reductant titanium (III) citrate and hydroxocobalamin are provided. When incubated with [methyl-13C] cobalamin and titanium citrate, both [methyl-13C] and unlabeled N-acetylphosphinothricin are produced. Our results suggest that SD_1168 catalyzes P-methylation using radical SAM-dependent chemistry with cobalamin as a coenzyme. In light of recent genomic information, the discovery of this P-methyltransferase suggests that S. denitrificans produces a phosphinate natural product.
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影响因子：
3.9
作者：
Allen, Kylie D.;Wang, Susan C.
通讯作者：
Wang, Susan C.
影响因子：
2.9
作者：
Gill, HS;Eisenberg, D
通讯作者：
Eisenberg, D
DOI：
10.1006/bbrc.2000.2313
发表时间：
2000-03-16
影响因子：
3.1
作者：
Broderick, JB;Henshaw, TF;Broderick, WE
通讯作者：
Broderick, WE
影响因子：
2.9
作者：
Cicchillo, RM;Iwig, DF;Booker, SJ
通讯作者：
Booker, SJ
影响因子：
3.5
作者：
Duschene KS;Broderick JB
通讯作者：
Broderick JB