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Purification and Physical—Chemical Properties of Methanobactin: A Chalkophore from Methylosinus trichosporium OB3b

Lookup NU author(s): Professor David Graham

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Abstract

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 sequence: N-2-isopropylester–(4-thionyl-5-hydroxy-imidazolate)–Gly1–Ser2–Cys3–Tyr4–pyrrolidine–(4-hydroxy-5-thionyl-imidazolate)–Ser5–Cys6–Met7 [Kim et al., (2004) Science 305, 1612-1615]. We report methods to purify methanobactin from M. trichosporium OB3b and present initial evidence of its physiological function. MALDI-TOF MS was used to systematically monitor samples to optimize purification conditions, and to detect and analyze 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 388 nm, a shoulder at 342 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 (maxex at 388 nm) and at 390 - 430 nm (maxex = 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.


Publication metadata

Author(s): Kim HJ, Galeva N, Larive CK, Alterman M, Graham DW

Publication type: Article

Publication status: Published

Journal: Biochemistry

Year: 2005

Volume: 44

Issue: 13

Pages: 5140-5148

Print publication date: 05/04/2005

ISSN (print): 0006-2960

ISSN (electronic): 1943-295X

Publisher: American Chemical Society

URL: http://dx.doi.org/10.1021/bi047367r

DOI: 10.1021/bi047367r


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