Alderwick, Luke J., Dover, Lynn G., Veerapen, Natacha, Gurcha, Sudagar S., Kremer, Laurent, Roper, David I., Pathak, Ashish K., Reynolds, Robert C., 1955- and Besra, Gurdyal S.. (2008) Expression, purification and characterisation of soluble GlfT and the identification of a novel galactofuranosyltransferase Rv3782 involved in priming GlfT-mediated galactan polymerisation in Mycobacterium tuberculosis. Protein Expression and Purification, Vol.58 (No.2). pp. 332-341. ISSN 1046-5928

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Abstract

The arabinogalactan (AG) component of the mycobacterial cell wall is an essential branched polysaccharide which tethers mycolic acids (m) to peptidoglycan (P), forming the mAGP complex. Much interest has been focused on the biosynthetic machinery involved in the production of this highly impermeable shield, which is the target for numerous anti-tuberculosis agents. The galactan domain of AG is synthesised via a bifunctional galactofuranosyltransferase (GUT), which utilises UDP-Galf as its high-energy substrate. However, it has proven difficult to study the protein in its recombinant form due to difficulties in recovering pure soluble protein using standard expression systems. Herein, we describe the effects of glfT co-induction with a range of chaperone proteins, which resulted in an appreciable yield of soluble protein at 5 mg/L after a one-step purification procedure. We have shown that this purified enzyme transfers (C-14]Galf to a range of both beta(1 -> 5) and beta(1 -> 6) linked digalactofuranosyl neoglycolipid acceptors with a distinct preference for the latter. Ligand binding studies using intrinsic tryptophan fluorescence have provided supporting evidence for the apparent preference of this enzyme to bind the beta(1 -> 6) disaccharide acceptor. However, we could not detect binding or gal actofuranosyltransferase activity with an n-octyl beta-D-Gal-(1 -> 4)-alpha-L-Rha acceptor, which mimics the reducing terminus of galactan in the mycobacterial cell wall. Conversely, after an extensive bioinformatics analysis of the H37Rv genome, further cloning, expression and functional analysis of the Rv3792 open reading frame indicates that this protein affords galactofuranosyltransferase activity against such an acceptor and paves the way for a better understanding of galactan biosynthesis in Mycobacterium tuberculosis. (C) 2007 Elsevier Inc. All rights reserved.

Item Type:	Journal Article
Subjects:	Q Science > QD Chemistry Q Science > QR Microbiology
Divisions:	Faculty of Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH):	Mycobacterium, Tuberculosis, Bacterial cell walls, Arabinogalactan, Glycosyltransferases, Polymerization
Journal or Publication Title:	Protein Expression and Purification
Publisher:	Academic Press
ISSN:	1046-5928
Date:	April 2008
Volume:	Vol.58
Number:	No.2
Number of Pages:	10
Page Range:	pp. 332-341
Identification Number:	10.1016/j.pep.2007.11.012
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Funder:	Royal Society (Great Britain), Medical Research Council (Great Britain) (MRC), Wellcome Trust (London, England)
Grant number:	081569/2/06/2 (WT)
References:	[1] C. Dye, Global epidemiology of tuberculosis, Lancet 367 (2006) 938– 940. [2] L.Y. Gao, F. Laval, E.H. Lawson, R.K. Groger, A. Woodruff, J.H. Morisaki, J.S. Cox, M. Daffe, E.J. Brown, Requirement for kasB in Mycobacterium mycolic acid biosynthesis, cell wall impermeability and intracellular survival: implications for therapy, Mol. Microbiol. 49 (2003) 1547–1563. [3] A.E. Belanger, G.S. Besra, M.E. Ford, K. Mikusova, J.T. Belisle, P.J. Brennan, J.M. Inamine, The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol, Proc. Natl. Acad. Sci. USA 93 (1996) 11919–11924. [4] A. Banerjee, E. Dubnau, A. Quemard, V. Balasubramanian, K.S. Um, T. Wilson, D. Collins, G. de Lisle, W.R. Jacobs Jr., inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis, Science 263 (1994) 227–230. [5] K. Takayama, E.L. Armstrong, K.A. Kunugi, J.O. Kilburn, Inhibition by ethambutol of mycolic acid transfer into the cell wall of Mycobacterium smegmatis, Antimicrob. Agents Chemother. 16 (1979) 240–242. [6] K. Takayama, J.O. Kilburn, Inhibition of synthesis of arabinogalactan by ethambutol in Mycobacterium smegmatis, Antimicrob. Agents Chemother. 33 (1989) 1493–1499. [7] K. Mikusova, R.A. Slayden, G.S. Besra, P.J. Brennan, Biogenesis of the mycobacterial cell wall and the site of action of ethambutol, Antimicrob. Agents Chemother. 39 (1995) 2484–2489. [8] A. Quemard, C. Lacave, G. Laneelle, Isoniazid inhibition of mycolic acid synthesis by cell extracts of sensitive and resistant strains of Mycobacterium aurum, Antimicrob. Agents Chemother. 35 (1991) 1035–1039. [9] M.H. Larsen, C. Vilcheze, L. Kremer, G.S. Besra, L. Parsons, M. Salfinger, L. Heifets, M.H. Hazbon, D. Alland, J.C. Sacchettini, W.R. Jacobs Jr., Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis, Mol. Microbiol. 46 (2002) 453–466. [10] J.O. Kilburn, K. Takayama, Effects of ethambutol on accumulation and secretion of trehalose mycolates and free mycolic acid in Mycobacterium smegmatis, Antimicrob. Agents Chemother. 20 (1981) 401–404. [11] E. Radmacher, K.C. Stansen, G.S. Besra, L.J. Alderwick, W.N. Maughan, G. Hollweg, H. Sahm, V.F. Wendisch, L. Eggeling, Ethambutol, a cell wall inhibitor of Mycobacterium tuberculosis, elicits L-glutamate efflux of Corynebacterium glutamicum, Microbiology 151 (2005) 1359–1368. [12] L.J. Alderwick, E. Radmacher, M. Seidel, R. Gande, P.G. Hitchen, H.R. Morris, A. Dell, H. Sahm, L. Eggeling, G.S. Besra, Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core, J. Biol. Chem. 280 (2005) 32362–32371. [13] V.E. Escuyer, M.A. Lety, J.B. Torrelles, K.H. Khoo, J.B. Tang, C.D. Rithner, C. Frehel, M.R. McNeil, P.J. Brennan, D. Chatterjee, The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan, J. Biol. Chem. 276 (2001) 48854–48862. [14] G.S. Besra, K.H. Khoo, M.R. McNeil, A. Dell, H.R. Morris, P.J. Brennan, A new interpretation of the structure of the mycolylarabinogalactan complex of Mycobacterium tuberculosis as revealed through characterization of oligoglycosylalditol fragments by fastatom bombardment mass spectrometry and 1H nuclear magnetic resonance spectroscopy, Biochemistry 34 (1995) 4257–4266. [15] M. McNeil, M. Daffe, P.J. Brennan, Evidence for the nature of the link between the arabinogalactan and peptidoglycan of mycobacterial cell walls, J. Biol. Chem. 265 (1990) 18200–18206. [16] M. Daffe, P.J. Brennan, M. McNeil, Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C NMR analyses, J. Biol. Chem. 265 (1990) 6734–6743. [17] I.C. Hancock, S. Carman, G.S. Besra, P.J. Brennan, E. Waite, Ligation of arabinogalactan to peptidoglycan in the cell wall of Mycobacterium smegmatis requires concomitant synthesis of the two wall polymers, Microbiology 148 (2002) 3059–3067. [18] K. Mikusova, M. Mikus, G.S. Besra, I. Hancock, P.J. Brennan, Biosynthesis of the linkage region of the mycobacterial cell wall, J. Biol. Chem. 271 (1996) 7820–7828. [19] K. Mikusova, T. Yagi, R. Stern, M.R. McNeil, G.S. Besra, D.C. Crick, P.J. Brennan, Biosynthesis of the galactan component of the mycobacterial cell wall, J. Biol. Chem. 275 (2000) 33890–33897. [20] L.J. Alderwick, M. Seidel, H. Sahm, G.S. Besra, L. Eggeling, Identification of a novel arabinofuranosyltransferase (AftA) involved in cell wall arabinan biosynthesis in Mycobacterium tuberculosis, J. Biol. Chem. 281 (2006) 15653–15661. [21] J.A. Mills, K. Motichka, M. Jucker, H.P. Wu, B.C. Uhlik, R.J. Stern, M.S. Scherman, V.D. Vissa, F. Pan, M. Kundu, Y.F. Ma, M. McNeil, Inactivation of the mycobacterial rhamnosyltransferase, which is needed for the formation of the arabinogalactan–peptidoglycan linker, leads to irreversible loss of viability, J. Biol. Chem. 279 (2004) 43540–43546. [22] L. Kremer, L.G. Dover, C. Morehouse, P. Hitchin, M. Everett, H.R. Morris, A. Dell, P.J. Brennan, M.R. McNeil, C. Flaherty, K. Duncan, G.S. Besra, Galactan biosynthesis in Mycobacterium tuberculosis. Identification of a bifunctional UDP-galactofuranosyltransferase, J. Biol. Chem. 276 (2001) 26430–26440. [23] K. Nishihara, M. Kanemori, M. Kitagawa, H. Yanagi, T. Yura, Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli, Appl. Environ. Microbiol. 64 (1998) 1694–1699. [24] K. Nishihara, M. Kanemori, H. Yanagi, T. Yura, Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli, Appl. Environ. Microbiol. 66 (2000) 884–889. [25] K. Mikusova, M. Belanova, J. Kordulakova, K. Honda, M.R. McNeil, S. Mahapatra, D.C. Crick, P.J. Brennan, Identification of a novel galactosyl transferase involved in biosynthesis of the mycobacterial cell wall, J. Bacteriol. 188 (2006) 6592–6598. [26] D. Hanahan, Studies on transformation of Escherichia coli with plasmids, J. Mol. Biol. 166 (1983) 557–580. [27] A.K. Pathak, V. Pathak, L. Seitz, J.A. Maddry, S.S. Gurcha, G.S. Besra, W.J. Suling, R.C. Reynolds, Studies on (beta, 1fi5) and (beta, 1fi6) linked octyl Gal(f) disaccharides as substrates for mycobacterial galactosyltransferase activity, Bioorg. Med. Chem. 9 (2001) 3129– 3143. [28] A.K. Pathak, G.S. Besra, D. Crick, J.A. Maddry, C.B. Morehouse, W.J. Suling, R.C. Reynolds, Studies on beta-D-Gal(f)- (1fi4)-alpha-L-Rha(p) octyl analogues as substrates for mycobacterial galactosyl transferase activity, Bioorg. Med. Chem. 7 (1999) 2407–2413. [29] R. Koplin, J.R. Brisson, C. Whitfield, UDP-galactofuranose precursor required for formation of the lipopolysaccharide O antigen of Klebsiella pneumoniae serotype O1 is synthesized by the product of the rfbDKPO1 gene, J. Biol. Chem. 272 (1997) 4121–4128. [30] Y.E. Tsvetkov, A.V. Nikolaev, The first chemical synthesis of UDP-a- D-galactofuranose, J. Chem. Soc., Perkin Trans. 1 (2000) 889–891. [31] N.L. Rose, G.C. Completo, S.J. Lin, M. McNeil, M.M. Palcic, T.L. Lowary, Expression, purification, and characterization of a galactofuranosyltransferase involved in Mycobacterium tuberculosis arabinogalactan biosynthesis, J. Am. Chem. Soc. 128 (2006) 6721–6729. [32] Y. Ma, F. Pan, M. McNeil, Formation of dTDP-rhamnose is essential for growth of mycobacteria, J. Bacteriol. 184 (2002) 3392– 3395. [33] Y. Ma, J.A. Mills, J.T. Belisle, V. Vissa, M. Howell, K. Bowlin, M.S. Scherman, M. McNeil, Determination of the pathway for rhamnose biosynthesis in mycobacteria: cloning, sequencing and expression of the Mycobacterium tuberculosis gene encoding alpha-D-glucose-1- phosphate thymidylyltransferase, Microbiology 143 (Pt. 3) (1997) 937–945. [34] S.S. Gurcha, A.R. Baulard, L. Kremer, C. Locht, D.B. Moody, W. Muhlecker, C.E. Costello, D.C. Crick, P.J. Brennan, G.S. Besra, Ppm1, a novel polyprenol monophosphomannose synthase from Mycobacterium tuberculosis, Biochem. J. 365 (2002) 441–450. [35] B.C. VanderVen, J.D. Harder, D.C. Crick, J.T. Belisle, Exportmediated assembly of mycobacterial glycoproteins parallels eukaryotic pathways, Science 309 (2005) 941–943. [36] A.E. Belanger, J.M. Inamine, Genetics of cell wall biosynthesis, in: G.F. Hatfull, W.R. JacobsJr. (Eds.), Molecular Genetics of Mycobacteria, ASM Press, Washington, DC, 2000. [37] L.G. Dover, A.M. Cerdeno-Tarraga, M.J. Pallen, J. Parkhill, G.S. Besra, Comparative cell wall core biosynthesis in the mycolated pathogens, Mycobacterium tuberculosis and Corynebacterium diphtheriae, FEMS Microbiol. Rev. 28 (2004) 225–250. [38] M. Braibant, P. Gilot, J. Content, The ATP binding cassette (ABC) transport systems of Mycobacterium tuberculosis, FEMS Microbiol. Rev. 24 (2000) 449–467. [39] M.R. McNeil, K.G. Robuck, M. Harter, P.J. Brennan, Enzymatic evidence for the presence of a critical terminal hexa-arabinoside in the cell walls of Mycobacterium tuberculosis, Glycobiology 4 (1994) 165– 173.
URI:	http://wrap.warwick.ac.uk/id/eprint/30198

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