Purification and physical-chemical properties of methanobactin:: A chalkophore from Methylosinus trichosporium OB3b
Purification and physical-chemical properties of methanobactin:: A chalkophore from Methylosinus trichosporium OB3b
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DOI：
10.1021/bi047367r
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发表时间：
2005-04-05
期刊：
影响因子：
2.9
通讯作者：
Graham, DW
中科院分区：
文献类型：
--
作者：
Kim, HJ;Galeva, N;Graham, DW
甲烷结核蛋白是一种胞外与铜结合的色肽，来自甲烷氧化菌--三孢甲基杆菌OB3b，被认为参与了铜的解毒、封存和吸收。虽然甲烷结核蛋白很小(1217.2 Da)，但它具有复杂的三维大环结构，具有几个不寻常的部分。该分子与一个铜结合，并具有N-2-isopropylester-(4-thionyl-5-hydroxyimidazolate)-Gly(1)-Ser(2)-Cys(3)-Tyr(4)-pyrrolidine-(4-hydroxy-5-thionylimidazolate)-Ser(5)-CyS6-Met(7)序列[Kim，H.J.，et al.(2004)科学305,1612-1615]。我们报道了从毛霉菌OB3b中提纯甲烷菌素的方法，并提供了其生理功能的初步证据。利用MALDI-TOF MS对样品进行系统监测，以优化纯化条件，并检测和分析特定的金属-甲烷结核蛋白络合物。首先用铜稳定萃取物，然后在中性pH缓冲液中用反相高效液相色谱分离。纯化的甲基丁酸乙酯在342 nm处有最大吸收峰，在388 nm处有一个肩峰，在282 nm处有一个宽峰。这些特征在CuCl2滴定后消失，在335、356、290和255 nm处出现新特征。此外，甲烷结核还含有两个荧光基团，它们分别在440-460 nm(388 Nm)和390-430 nm(λ(Ex)(Max)=342 nm)处有较宽的发射。最后，当培养物暴露在较高的铜水平下时，甲烷菌素消除了青霉OB3b的生长滞后，并显著提高了生长速度。
Methanobactin is an extracellular, copper-binding chromopeptide from the methane-oxidizing bacterium, Methylosinus trichosporium OB3b, believed to be involved in copper detoxification, sequestration, and uptake. Although small (1217.2 Da), methanobactin possesses a complex three-dimensional macrocyclic structure with several unusual moieties. The molecule binds one copper and has the N-2-isopropylester-(4-thionyl-5-hydroxyimidazolate)-Gly(1)-Ser(2)-Cys(3)-Tyr(4)-pyrrolidine-(4-hydroxy-5-thionylimidazolate)-Ser(5)-CyS6-Met(7) sequence [Kim, H. J., et al. (2004) Science 305, 1612-1615]. We report methods for purifying methanobactin from M. trichosporium OB3b and present initial evidence of its physiological function. MALDI-TOF MS was used to systematically monitor samples for optimizing purification conditions, and for detecting and analyzing specific metal-methanobactin complexes. Purification was performed by first stabilizing the extracted compound with copper followed by separation using reversed-phase HPLC in neutral pH buffers. Purified methanobactin exhibited UV-visible maxima at 342 nm, a shoulder at 388 nm, and a broad peak at 282 nm. These features were lost upon CuCl2 titration with appearance of new features at 335, 356, 290, and 255 nm. Furthermore, methanobactin contains two fluorescent moieties, which exhibit broad emissions at 440-460 nm (lambda(ex)(max) at 388 nm) and 390-430 nm (lambda(ex)(max) = 342 nm), respectively. Finally, methanobactin eliminates the growth lag in M. trichosporium OB3b and substantially increases growth rates when cultures are exposed to elevated copper levels.