Expression, purification, and characterization of a galactofuranosyltransferase involved in Mycobacterium tuberculosis arabinogalactan biosynthesis.
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 microM 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.
Rose
Natisha L
NL
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.
Completo
Gladys C
GC
Lin
Shuang-Jun
SJ
McNeil
Michael
M
Palcic
Monica M
MM
Lowary
Todd L
TL
D019086:Bridged Bicyclo Compounds, Heterocyclic; D004926:Escherichia coli / Q000378:metabolism; D005685:Galactans / Q000096:biosynthesis*; D005700:Galactosyltransferases / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D007093:Imidazoles; D007700:Kinetics; D013058:Mass Spectrometry; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D013379:Substrate Specificity
D019086:Bridged Bicyclo Compounds, Heterocyclic; D005685:Galactans; D007093:Imidazoles; C095121:GLG-V 13; D005700:Galactosyltransferases; C005653:arabinogalactan