4.8 Article

Expression, purification, and characterization of a galactofuranosyltransferase involved in Mycobacterium tuberculosis arabinogalactan biosynthesis

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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 128, 期 20, 页码 6721-6729

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AMER CHEMICAL SOC
DOI: 10.1021/ja058254d

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  1. NIAID NIH HHS [1P01AI057836, AI 33706] Funding Source: Medline

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The major structural component of the cell wall of Mycobacterium tuberculosis is a lipidated polysaccharide, the mycoyl-arabinogalactan-peptidoglycan (mAGP) complex. This glycoconjugate plays a key role in the survival of the organism, and thus, enzymes involved in its biosynthesis have attracted attention as sites for drug action. At the core of the mAGP is a galactan composed of D-galactofuranose residues attached via alternating, beta-(1 -> 5) and beta( 1 -> 6) linkages. A single enzyme, glfT, has been shown to synthesize both glycosidic linkages. We report here the first high- level expression and purification of glfT by expression of the Rv3808c gene in Escherichia coli C41(DE3). Following a three- step purification procedure, 3-7 mg of protein of > 95% purity was isolated from each liter of culture. We subsequently probed the substrate specificity of glfT by evaluating a panel of potential mono- and oligosaccharide substrates and demonstrated, for the first time, that trisaccharides are better substrates than disaccharides and that one disaccharide, in which the terminal D-galactofuranose residue is replaced with an L-arabinofuranose moiety, is a weak substrate. Kinetic characterization of the enzyme using four of the oligosaccharide acceptors gave K-m values ranging from 204 mu M to 1.7 mM. Through the use of NMR spectroscopy and mass spectrometry, we demonstrated that this recombinant enzyme, like the wild-type protein, is bifunctional and can synthesize both beta-(1 -> 6) and beta-(1 -> 5)-linkages in an alternating fashion. Access to purified glfT is expected to facilitate the development of high-throughput assays for the identification of inhibitors of the enzyme, which are potential antituberculosis agents.

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