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W2031964267 abstract "The structural core of the cell walls ofMycobacterium spp. consists of peptidoglycan bound by a linker unit (-α-L-Rhap-(1→3)-D-GlcNAc-P-) to a galactofuran, which in turn is attached to arabinofuran and mycolic acids. The sequence of reactions leading to the biogenesis of this complex starts with the formation of the linker unit on a polyprenyl-P to produce polyprenyl-P-P-GlcNAc-Rha (Mikušová, K., Mikuš, M., Besra, G. S., Hancock, I., and Brennan, P. J. (1996) J. Biol. Chem. 271, 7820–7828). We now establish that formation of the galactofuran takes place on this intermediate with UDP-Galf as the Galf donor presented in the form of UDP-Galpand UDP-Galp mutase (the glf gene product) and is catalyzed by galactofuranosyl transferases, one of which, theMycobacterium tuberculosis H37Rv3808c gene product, has been identified. Evidence is also presented for the growth of the arabinofuran on this polyprenyl-P-P-linker unit-galactan intermediate catalyzed by unidentified arabinosyl transferases, with decaprenyl-P-Araf or 5-P-ribosyl-PP as the Arafdonor. The product of these steps, the lipid-linked-LU-galactan-arabinan has been partially characterized in terms of its heterogeneity, size, and composition. Biosynthesis of the major components of mycobacterial cell walls is proving to be extremely complex. However, partial definition of arabinogalactan synthesis, the site of action of several major anti-tuberculosis drugs, facilitates the present day thrust for new drugs to counteract multiple drug-resistant tuberculosis. The structural core of the cell walls ofMycobacterium spp. consists of peptidoglycan bound by a linker unit (-α-L-Rhap-(1→3)-D-GlcNAc-P-) to a galactofuran, which in turn is attached to arabinofuran and mycolic acids. The sequence of reactions leading to the biogenesis of this complex starts with the formation of the linker unit on a polyprenyl-P to produce polyprenyl-P-P-GlcNAc-Rha (Mikušová, K., Mikuš, M., Besra, G. S., Hancock, I., and Brennan, P. J. (1996) J. Biol. Chem. 271, 7820–7828). We now establish that formation of the galactofuran takes place on this intermediate with UDP-Galf as the Galf donor presented in the form of UDP-Galpand UDP-Galp mutase (the glf gene product) and is catalyzed by galactofuranosyl transferases, one of which, theMycobacterium tuberculosis H37Rv3808c gene product, has been identified. Evidence is also presented for the growth of the arabinofuran on this polyprenyl-P-P-linker unit-galactan intermediate catalyzed by unidentified arabinosyl transferases, with decaprenyl-P-Araf or 5-P-ribosyl-PP as the Arafdonor. The product of these steps, the lipid-linked-LU-galactan-arabinan has been partially characterized in terms of its heterogeneity, size, and composition. Biosynthesis of the major components of mycobacterial cell walls is proving to be extremely complex. However, partial definition of arabinogalactan synthesis, the site of action of several major anti-tuberculosis drugs, facilitates the present day thrust for new drugs to counteract multiple drug-resistant tuberculosis. linker unit arabinogalactan high pressure liquid chromatography glycolipid β-D-arabinofuranosyl-1-monophosphoryl-decaprenol 4-morpholinepropanesulfonic acid polyacrylamide gel electrophoresis N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine The cell wall of Mycobacterium spp. is required for growth and survival (1Daffé M. Draper P. Adv. Microbiol. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar), and its formation has become the focus of the search for essential targets in the development of new drugs against tuberculosis (2Crick D.C. Brennan P.J. Curr. Opin. Anti-Inf. Invest. Drugs. 2000; 2: 154-163Google Scholar). Some of the constituents of the widely applied anti-tuberculosis four-drug regimen DOTS (directlyobserved therapy, short course) (isoniazid, ethionamide, and ethambutol) affect mycolic acid or arabinan biosynthesis (3Ramaswamy S. Musser J.M. Tubercle Lung Dis. 1998; 79: 3-29Abstract Full Text PDF PubMed Scopus (925) Google Scholar). The infrastructure, or core, of the cell wall of Mycobacterium tuberculosis is composed of a covalently linked complex of mycolic acids, arabinan, and galactan attached to peptidoglycan by a -Rhap(1→3)GlcNAc-P- linker unit (LU)1 (4McNeil M. Daffé M. Brennan P.J. J. Biol. Chem. 1990; 265: 18200-18206Abstract Full Text PDF PubMed Google Scholar), the mycolyl-AG-LU-peptidoglycan complex. Most of the primary structure has been elucidated (5Daffé M. Brennan P.J. McNeil M. J. Biol. Chem. 1990; 265: 6734-6743Abstract Full Text PDF PubMed Google Scholar), and new evidence supports the concept of a dynamic, asymmetric, lipid bilayer in which the mycolic acid monolayer, interspersed with porin-like proteins and perpendicular to the arabinogalactan-peptidoglycan complex, is complemented by an assortment of phospholipids and glycolipids to provide a relatively impermeable lipid barrier (6Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1545) Google Scholar).The initial steps in the assembly of the complex have been recently defined (7Mikušová K. Mikuš M. Besra G.S. Hancock I. Brennan P.J. J. Biol. Chem. 1996; 271: 7820-7828Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Isolated membranes and cell walls from Mycobacterium smegmatis catalyze the transfer of GlcNAc-1-P and Rha from their respective nucleotide donors to endogenous polyprenyl-P (probably C50-P), giving rise to polyprenyl-P-P-GlcNAc (GL-1) and polyprenyl-P-P-GlcNAc-Rha (GL-2), followed by the addition of two successive Galf units, donated by UDP-Galp to give rise to polyprenyl-P-P-GlcNAc-Rha-Galf (GL-3) and polyprenyl-P-P-GlcNAc-Rha-(Galf)2 (GL-4). It has since been shown that UDP-Galp is the direct precursor of UDP-Galf, catalyzed by UDP-Galp mutase (theglf gene product) (8Weston A. Stern R.J. Lee R.E. Nassau P.M. Monsey D. Marin S.L. Scherman M.S. Besra G.S. Duncan K. McNeil M.R. Tubercle Lung Dis. 1998; 78: 123-131Abstract Full Text PDF Scopus (103) Google Scholar). We now establish that the enzymes responsible for the conversion of UDP-Galp to the Galf-containing GL-3, GL-4, etc. and the polyprenol-P-linked galactan are UDP-Galp mutase and galactofuranosyl transferase(s), one of which, M. tuberculosisH37Rv3808c, was identified. We demonstrate that Araf is also transferred to the polyprenol-P-linked galactan, catalyzed by unidentified membrane-cell wall-associated arabinofuranosyl transferases using either decaprenyl-P-Ara or P-ribosyl-PP as donor. The polyprenyl-P-P LU-galactan-arabinan has been partially characterized in terms of its heterogeneity, size, and linkage, and evidence for the presence of each entity is presented. The work provides the first insights into the complexity of arabinogalactan synthesis in Mycobacterium, which apparently occurs in conjunction with de novo mycolic acid synthesis and mycolyl group transfer and final ligation of the complex to peptidoglycan.DISCUSSIONTo date, the only polyprenyl-P implicated in aspects of mycobacterial cell wall biosynthesis are decaprenyl-P and heptaprenyl-P (27Takayama K. Schnoes H.K. Semmler E.J. Biochim. Biophys. Acta. 1973; 136: 217-221Google Scholar, 28Besra G.S. Morehouse C.B. Rittner C.M. Waechter C.J. Brennan P.J. J. Biol. Chem. 1997; 272: 18460-18466Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The addition of a cell wall-membrane enzyme preparation and UDP-[14C]Galp to reaction mixtures capable of synthesizing polyprenyl-P-P-GlcNAc (GL-1) and polyprenyl-P-P-GlcNAc-Rha (GL-2) resulted in the synthesis of Galf-labeled more polar glycolipids, GL-3 and GL-4, indicating stepwise growth of the initial segments of the galactan chain on the polyprenyl-P-P-GlcNAc-Rha unit, 1 Gal unit at a time (7Mikušová K. Mikuš M. Besra G.S. Hancock I. Brennan P.J. J. Biol. Chem. 1996; 271: 7820-7828Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Present evidence shows that thoroughly washed membrane preparations are not able to synthesize GL-3 and GL-4, which, however, could be achieved by the addition of UDP-Galpmutase encoded by the glf gene (11Ma Y. Mills J.A. Belisle J.T. Vissa V. Howell M. Bowlin K. Scherman M.S. McNeil M. Microbiology. 1997; 143: 937-945Crossref PubMed Scopus (58) Google Scholar), demonstrating a requirement for UDP-Galf as donor. Moreover, analysis of the CHCl3/CH3OH (2:1)-soluble lipids in polar solvent demonstrated a hierarchial array of galactolipids, with all of the evidence for polyprenyl-P linkage, again pointing to sequential addition of single Galf units. Calling on the approaches that led to the solubilization of the oligosaccharide-P-P-dolichol intermediates of glycoprotein synthesis (13Rush J.S. Shelling J.G. Zingg N.S. Ray P.H. Waechter C.J. J. Biol. Chem. 1993; 268: 13110-13117Abstract Full Text PDF PubMed Google Scholar) and to the extraction of phosphosphingolipids from yeast (14Angus W.W. Lester R.L. Arch. Biochem. Biophys. 1972; 151: 483-495Crossref PubMed Scopus (68) Google Scholar) and the lipophosphoglycan of Leishmania donovani (29McNeely T.B. Turco S.J. J. Immunol. 1990; 144: 2745-2750PubMed Google Scholar), we successfully solubilized the newly synthesized galactofuran. Surprisingly, two distinct populations exist, differentially extracted by the two solvents. As in the case of the dolichyl-bound oligosaccharides, the identification of a polyprenol-P linkage was based on mild acid lability, mild alkali stability, solubility in extremely polar organic solvents, and exclusion from Bio-Gel P-100, all suggesting a highly polymerized lipid-linked version of GL 1–4.De novo synthesis of the lipid-linked polymer is also sensitive to tunicamycin, and evidence is presented for the incorporation of GL-1/GL-2 into the lipid-linked polymer. Glycosyl linkage analysis of the polymer produced t-Galf, 5-linked Galf, 6-linked Galf, and 5,6-linked Galf, indicating that there is substitution of one or more of the linear Galf residues, presumably with arabinan. Moreover, [14C]Araf donated by synthetic C50-P-[14C]Araf, or formed from 5-phospho-[14C]ribosyl-pyrophosphate was incorporated into this same polymer as characterized by solubility in polar lipid solvents, SDS-PAGE mobility, mild acid lability, and hence lipid linkage. The combined evidence points to the pathway shown in Fig.9 for the synthesis of the AG component of the mycolylarabinogalactan complex of mycobacterial cell walls. Clearly, this cell-free system does not allow for significant transfer of the newly synthesized AG from polyprenyl-P-P to peptidoglycan, in that relatively little of the [14C]Gal from UDP-[14C]Gal ends up in the insoluble mycolylarabinogalactan.The search for galactosyl transferases responsible for this galactan AG elongation through comparisons with various families of galactopyranosyl transferases (30Breton C. Bettler E. Joziasse D.H. Geremia R.A. Imberty A. J. Biochem. 1998; 123: 1000-1009Crossref PubMed Scopus (136) Google Scholar) proved to be uninformative. However, analysis of M. tuberculosis Rv3808c, which is linked directly to the glf gene (Rv3809c), showed strong indications of a glycosyl transferase. Alignment of the hydrophobic cluster analysis plot (31Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (541) Google Scholar) of the predicted amino acid sequence ofM. tuberculosis Rv3808c with plots of other known β-glycosyltransferases (26Saxena I.M. Brown Jr., R.M. Fevre M. Geremia R.A. Henrissat B. J. Bacteriol. 1995; 177: 1419-1424Crossref PubMed Google Scholar) revealed a common domain structure of repeating α-helix and β-strand motifs between amino acids 161 and 262, corresponding to domain A of glycosyltransferases (31Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (541) Google Scholar). Within this domain, two aspartic acid residues at 199 and 256 had the hallmarks of highly conserved residues within the C-terminal loops of the β-2 and β-4 strands, the characteristic signature of all β-glycosyltransferases (26Saxena I.M. Brown Jr., R.M. Fevre M. Geremia R.A. Henrissat B. J. Bacteriol. 1995; 177: 1419-1424Crossref PubMed Google Scholar). There was no evidence of domain B in Rv3808c (and no conserved QXXRW amino acid motif) characteristic of β-glycosyltransferases that add sugars processively to the reducing end of a polysaccharide chain. β-Glycosyltransferases that add sugar residues to the nonreducing end of the polysaccharide chain have only the one domain, A (26Saxena I.M. Brown Jr., R.M. Fevre M. Geremia R.A. Henrissat B. J. Bacteriol. 1995; 177: 1419-1424Crossref PubMed Google Scholar). Thus, the synthesis of mycobacterial galactan may share similarities with biosynthesis of the homopolymer O-antigenic d-galactans of E. coliO8 and O9 and Klebsiella pneumoniae O1. In both cases, synthesis is initiated by the transfer of GlcNAc-P to polyprenyl-P (32Whitfield C. Trends Microbiol. 1995; 3: 178-185Abstract Full Text PDF PubMed Scopus (266) Google Scholar), and, during formation of the E. coli O9 antigen, mannosyl residues are rapidly added to the nonreducing terminus of the acceptor one residue at a time, in a processive mechanism (33Lind T. Lindhal U. Lindholt K. J. Biol. Chem. 1993; 268: 20705-20708Abstract Full Text PDF PubMed Google Scholar).d-Galactan I from K. pneumoniae is assembled on the cytoplasmic face of the plasma membrane, and polymerization is thought to occur by sequential sugar transfer on the lipid intermediate. An ATP-binding cassette (ABC) transporter then translocates polymerized D-galactan I across the plasma membrane prior to ligation to lipid A core (33Lind T. Lindhal U. Lindholt K. J. Biol. Chem. 1993; 268: 20705-20708Abstract Full Text PDF PubMed Google Scholar).The elucidation of the basic elements of synthesis of the cell wall core of mycobacteria should substantially enhance current tuberculosis drug discovery efforts, in that aspects of cell wall synthesis are the targets of many of the current front-line anti-tuberculosis drugs (2Crick D.C. Brennan P.J. Curr. Opin. Anti-Inf. Invest. Drugs. 2000; 2: 154-163Google Scholar), and the pathways and their end products are distinctly xenogeneic. The cell wall of Mycobacterium spp. is required for growth and survival (1Daffé M. Draper P. Adv. Microbiol. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar), and its formation has become the focus of the search for essential targets in the development of new drugs against tuberculosis (2Crick D.C. Brennan P.J. Curr. Opin. Anti-Inf. Invest. Drugs. 2000; 2: 154-163Google Scholar). Some of the constituents of the widely applied anti-tuberculosis four-drug regimen DOTS (directlyobserved therapy, short course) (isoniazid, ethionamide, and ethambutol) affect mycolic acid or arabinan biosynthesis (3Ramaswamy S. Musser J.M. Tubercle Lung Dis. 1998; 79: 3-29Abstract Full Text PDF PubMed Scopus (925) Google Scholar). The infrastructure, or core, of the cell wall of Mycobacterium tuberculosis is composed of a covalently linked complex of mycolic acids, arabinan, and galactan attached to peptidoglycan by a -Rhap(1→3)GlcNAc-P- linker unit (LU)1 (4McNeil M. Daffé M. Brennan P.J. J. Biol. Chem. 1990; 265: 18200-18206Abstract Full Text PDF PubMed Google Scholar), the mycolyl-AG-LU-peptidoglycan complex. Most of the primary structure has been elucidated (5Daffé M. Brennan P.J. McNeil M. J. Biol. Chem. 1990; 265: 6734-6743Abstract Full Text PDF PubMed Google Scholar), and new evidence supports the concept of a dynamic, asymmetric, lipid bilayer in which the mycolic acid monolayer, interspersed with porin-like proteins and perpendicular to the arabinogalactan-peptidoglycan complex, is complemented by an assortment of phospholipids and glycolipids to provide a relatively impermeable lipid barrier (6Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1545) Google Scholar). The initial steps in the assembly of the complex have been recently defined (7Mikušová K. Mikuš M. Besra G.S. Hancock I. Brennan P.J. J. Biol. Chem. 1996; 271: 7820-7828Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Isolated membranes and cell walls from Mycobacterium smegmatis catalyze the transfer of GlcNAc-1-P and Rha from their respective nucleotide donors to endogenous polyprenyl-P (probably C50-P), giving rise to polyprenyl-P-P-GlcNAc (GL-1) and polyprenyl-P-P-GlcNAc-Rha (GL-2), followed by the addition of two successive Galf units, donated by UDP-Galp to give rise to polyprenyl-P-P-GlcNAc-Rha-Galf (GL-3) and polyprenyl-P-P-GlcNAc-Rha-(Galf)2 (GL-4). It has since been shown that UDP-Galp is the direct precursor of UDP-Galf, catalyzed by UDP-Galp mutase (theglf gene product) (8Weston A. Stern R.J. Lee R.E. Nassau P.M. Monsey D. Marin S.L. Scherman M.S. Besra G.S. Duncan K. McNeil M.R. Tubercle Lung Dis. 1998; 78: 123-131Abstract Full Text PDF Scopus (103) Google Scholar). We now establish that the enzymes responsible for the conversion of UDP-Galp to the Galf-containing GL-3, GL-4, etc. and the polyprenol-P-linked galactan are UDP-Galp mutase and galactofuranosyl transferase(s), one of which, M. tuberculosisH37Rv3808c, was identified. We demonstrate that Araf is also transferred to the polyprenol-P-linked galactan, catalyzed by unidentified membrane-cell wall-associated arabinofuranosyl transferases using either decaprenyl-P-Ara or P-ribosyl-PP as donor. The polyprenyl-P-P LU-galactan-arabinan has been partially characterized in terms of its heterogeneity, size, and linkage, and evidence for the presence of each entity is presented. The work provides the first insights into the complexity of arabinogalactan synthesis in Mycobacterium, which apparently occurs in conjunction with de novo mycolic acid synthesis and mycolyl group transfer and final ligation of the complex to peptidoglycan. DISCUSSIONTo date, the only polyprenyl-P implicated in aspects of mycobacterial cell wall biosynthesis are decaprenyl-P and heptaprenyl-P (27Takayama K. Schnoes H.K. Semmler E.J. Biochim. Biophys. Acta. 1973; 136: 217-221Google Scholar, 28Besra G.S. Morehouse C.B. Rittner C.M. Waechter C.J. Brennan P.J. J. Biol. Chem. 1997; 272: 18460-18466Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The addition of a cell wall-membrane enzyme preparation and UDP-[14C]Galp to reaction mixtures capable of synthesizing polyprenyl-P-P-GlcNAc (GL-1) and polyprenyl-P-P-GlcNAc-Rha (GL-2) resulted in the synthesis of Galf-labeled more polar glycolipids, GL-3 and GL-4, indicating stepwise growth of the initial segments of the galactan chain on the polyprenyl-P-P-GlcNAc-Rha unit, 1 Gal unit at a time (7Mikušová K. Mikuš M. Besra G.S. Hancock I. Brennan P.J. J. Biol. Chem. 1996; 271: 7820-7828Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Present evidence shows that thoroughly washed membrane preparations are not able to synthesize GL-3 and GL-4, which, however, could be achieved by the addition of UDP-Galpmutase encoded by the glf gene (11Ma Y. Mills J.A. Belisle J.T. Vissa V. Howell M. Bowlin K. Scherman M.S. McNeil M. Microbiology. 1997; 143: 937-945Crossref PubMed Scopus (58) Google Scholar), demonstrating a requirement for UDP-Galf as donor. Moreover, analysis of the CHCl3/CH3OH (2:1)-soluble lipids in polar solvent demonstrated a hierarchial array of galactolipids, with all of the evidence for polyprenyl-P linkage, again pointing to sequential addition of single Galf units. Calling on the approaches that led to the solubilization of the oligosaccharide-P-P-dolichol intermediates of glycoprotein synthesis (13Rush J.S. Shelling J.G. Zingg N.S. Ray P.H. Waechter C.J. J. Biol. Chem. 1993; 268: 13110-13117Abstract Full Text PDF PubMed Google Scholar) and to the extraction of phosphosphingolipids from yeast (14Angus W.W. Lester R.L. Arch. Biochem. Biophys. 1972; 151: 483-495Crossref PubMed Scopus (68) Google Scholar) and the lipophosphoglycan of Leishmania donovani (29McNeely T.B. Turco S.J. J. Immunol. 1990; 144: 2745-2750PubMed Google Scholar), we successfully solubilized the newly synthesized galactofuran. Surprisingly, two distinct populations exist, differentially extracted by the two solvents. As in the case of the dolichyl-bound oligosaccharides, the identification of a polyprenol-P linkage was based on mild acid lability, mild alkali stability, solubility in extremely polar organic solvents, and exclusion from Bio-Gel P-100, all suggesting a highly polymerized lipid-linked version of GL 1–4.De novo synthesis of the lipid-linked polymer is also sensitive to tunicamycin, and evidence is presented for the incorporation of GL-1/GL-2 into the lipid-linked polymer. Glycosyl linkage analysis of the polymer produced t-Galf, 5-linked Galf, 6-linked Galf, and 5,6-linked Galf, indicating that there is substitution of one or more of the linear Galf residues, presumably with arabinan. Moreover, [14C]Araf donated by synthetic C50-P-[14C]Araf, or formed from 5-phospho-[14C]ribosyl-pyrophosphate was incorporated into this same polymer as characterized by solubility in polar lipid solvents, SDS-PAGE mobility, mild acid lability, and hence lipid linkage. The combined evidence points to the pathway shown in Fig.9 for the synthesis of the AG component of the mycolylarabinogalactan complex of mycobacterial cell walls. Clearly, this cell-free system does not allow for significant transfer of the newly synthesized AG from polyprenyl-P-P to peptidoglycan, in that relatively little of the [14C]Gal from UDP-[14C]Gal ends up in the insoluble mycolylarabinogalactan.The search for galactosyl transferases responsible for this galactan AG elongation through comparisons with various families of galactopyranosyl transferases (30Breton C. Bettler E. Joziasse D.H. Geremia R.A. Imberty A. J. Biochem. 1998; 123: 1000-1009Crossref PubMed Scopus (136) Google Scholar) proved to be uninformative. However, analysis of M. tuberculosis Rv3808c, which is linked directly to the glf gene (Rv3809c), showed strong indications of a glycosyl transferase. Alignment of the hydrophobic cluster analysis plot (31Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (541) Google Scholar) of the predicted amino acid sequence ofM. tuberculosis Rv3808c with plots of other known β-glycosyltransferases (26Saxena I.M. Brown Jr., R.M. Fevre M. Geremia R.A. Henrissat B. J. Bacteriol. 1995; 177: 1419-1424Crossref PubMed Google Scholar) revealed a common domain structure of repeating α-helix and β-strand motifs between amino acids 161 and 262, corresponding to domain A of glycosyltransferases (31Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (541) Google Scholar). Within this domain, two aspartic acid residues at 199 and 256 had the hallmarks of highly conserved residues within the C-terminal loops of the β-2 and β-4 strands, the characteristic signature of all β-glycosyltransferases (26Saxena I.M. Brown Jr., R.M. Fevre M. Geremia R.A. Henrissat B. J. Bacteriol. 1995; 177: 1419-1424Crossref PubMed Google Scholar). There was no evidence of domain B in Rv3808c (and no conserved QXXRW amino acid motif) characteristic of β-glycosyltransferases that add sugars processively to the reducing end of a polysaccharide chain. β-Glycosyltransferases that add sugar residues to the nonreducing end of the polysaccharide chain have only the one domain, A (26Saxena I.M. Brown Jr., R.M. Fevre M. Geremia R.A. Henrissat B. J. Bacteriol. 1995; 177: 1419-1424Crossref PubMed Google Scholar). Thus, the synthesis of mycobacterial galactan may share similarities with biosynthesis of the homopolymer O-antigenic d-galactans of E. coliO8 and O9 and Klebsiella pneumoniae O1. In both cases, synthesis is initiated by the transfer of GlcNAc-P to polyprenyl-P (32Whitfield C. Trends Microbiol. 1995; 3: 178-185Abstract Full Text PDF PubMed Scopus (266) Google Scholar), and, during formation of the E. coli O9 antigen, mannosyl residues are rapidly added to the nonreducing terminus of the acceptor one residue at a time, in a processive mechanism (33Lind T. Lindhal U. Lindholt K. J. Biol. Chem. 1993; 268: 20705-20708Abstract Full Text PDF PubMed Google Scholar).d-Galactan I from K. pneumoniae is assembled on the cytoplasmic face of the plasma membrane, and polymerization is thought to occur by sequential sugar transfer on the lipid intermediate. An ATP-binding cassette (ABC) transporter then translocates polymerized D-galactan I across the plasma membrane prior to ligation to lipid A core (33Lind T. Lindhal U. Lindholt K. J. Biol. Chem. 1993; 268: 20705-20708Abstract Full Text PDF PubMed Google Scholar).The elucidation of the basic elements of synthesis of the cell wall core of mycobacteria should substantially enhance current tuberculosis drug discovery efforts, in that aspects of cell wall synthesis are the targets of many of the current front-line anti-tuberculosis drugs (2Crick D.C. Brennan P.J. Curr. Opin. Anti-Inf. Invest. Drugs. 2000; 2: 154-163Google Scholar), and the pathways and their end products are distinctly xenogeneic. To date, the only polyprenyl-P implicated in aspects of mycobacterial cell wall biosynthesis are decaprenyl-P and heptaprenyl-P (27Takayama K. Schnoes H.K. Semmler E.J. Biochim. Biophys. Acta. 1973; 136: 217-221Google Scholar, 28Besra G.S. Morehouse C.B. Rittner C.M. Waechter C.J. Brennan P.J. J. Biol. Chem. 1997; 272: 18460-18466Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The addition of a cell wall-membrane enzyme preparation and UDP-[14C]Galp to reaction mixtures capable of synthesizing polyprenyl-P-P-GlcNAc (GL-1) and polyprenyl-P-P-GlcNAc-Rha (GL-2) resulted in the synthesis of Galf-labeled more polar glycolipids, GL-3 and GL-4, indicating stepwise growth of the initial segments of the galactan chain on the polyprenyl-P-P-GlcNAc-Rha unit, 1 Gal unit at a time (7Mikušová K. Mikuš M. Besra G.S. Hancock I. Brennan P.J. J. Biol. Chem. 1996; 271: 7820-7828Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Present evidence shows that thoroughly washed membrane preparations are not able to synthesize GL-3 and GL-4, which, however, could be achieved by the addition of UDP-Galpmutase encoded by the glf gene (11Ma Y. Mills J.A. Belisle J.T. Vissa V. Howell M. Bowlin K. Scherman M.S. McNeil M. Microbiology. 1997; 143: 937-945Crossref PubMed Scopus (58) Google Scholar), demonstrating a requirement for UDP-Galf as donor. Moreover, analysis of the CHCl3/CH3OH (2:1)-soluble lipids in polar solvent demonstrated a hierarchial array of galactolipids, with all of the evidence for polyprenyl-P linkage, again pointing to sequential addition of single Galf units. Calling on the approaches that led to the solubilization of the oligosaccharide-P-P-dolichol intermediates of glycoprotein synthesis (13Rush J.S. Shelling J.G. Zingg N.S. Ray P.H. Waechter C.J. J. Biol. Chem. 1993; 268: 13110-13117Abstract Full Text PDF PubMed Google Scholar) and to the extraction of phosphosphingolipids from yeast (14Angus W.W. Lester R.L. Arch. Biochem. Biophys. 1972; 151: 483-495Crossref PubMed Scopus (68) Google Scholar) and the lipophosphoglycan of Leishmania donovani (29McNeely T.B. Turco S.J. J. Immunol. 1990; 144: 2745-2750PubMed Google Scholar), we successfully solubilized the newly synthesized galactofuran. Surprisingly, two distinct populations exist, differentially extracted by the two solvents. As in the case of the dolichyl-bound oligosaccharides, the identification of a polyprenol-P linkage was based on mild acid lability, mild alkali stability, solubility in extremely polar organic solvents, and exclusion from Bio-Gel P-100, all suggesting a highly polymerized lipid-linked version of GL 1–4.De novo synthesis of the lipid-linked polymer is also sensitive to tunicamycin, and evidence is presented for the incorporation of GL-1/GL-2 into the lipid-linked polymer. Glycosyl linkage analysis of the polymer produced t-Galf, 5-linked Galf, 6-linked Galf, and 5,6-linked Galf, indicating that there is substitution of one or more of the linear Galf residues, presumably with arabinan. Moreover, [14C]Araf donated by synthetic C50-P-[14C]Araf, or formed from 5-phospho-[14C]ribosyl-pyrophosphate was incorporated into this same polymer as characterized by solubility in polar lipid solvents, SDS-PAGE mobility, mild acid lability, and hence lipid linkage. The combined evidence points to the pathway shown in Fig.9 for the synthesis of the AG component of the mycolylarabinogalactan complex of mycobacterial cell walls. Clearly, this cell-free system does not allow for significant transfer of the newly synthesized AG from polyprenyl-P-P to peptidoglycan, in that relatively little of the [14C]Gal from UDP-[14C]Gal ends up in the insoluble mycolylarabinogalactan. The search for galactosyl transferases responsible for this galactan AG elongation through comparisons with various families of galactopyranosyl transferases (30Breton C. Bettler E. Joziasse D.H. Geremia R.A. Imberty A. J. Biochem. 1998; 123: 1000-1009Crossref PubMed Scopus (136) Google Scholar) proved to be uninformative. However, analysis of M. tuberculosis Rv3808c, which is linked directly to the glf gene (Rv3809c), showed strong indications of a glycosyl transferase. Alignment of the hydrophobic cluster analysis plot (31Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (541) Google Scholar) of the predicted amino acid sequence ofM. tuberculosis Rv3808c with plots of other known β-glycosyltransferases (26Saxena I.M. Brown Jr., R.M. Fevre M. Geremia R.A. Henrissat B. J. Bacteriol. 1995; 177: 1419-1424Crossref PubMed Google Scholar) revealed a common domain structure of repeating α-helix and β-strand motifs between amino acids 161 and 262, corresponding to domain A of glycosyltransferases (31Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (541) Google Scholar). Within this domain, two aspartic acid residues at 199 and 256 had the hallmarks of highly conserved residues within the C-terminal loops of the β-2 and β-4 strands, the characteristic signature of all β-glycosyltransferases (26Saxena I.M. Brown Jr., R.M. Fevre M. Geremia R.A. Henrissat B. J. Bacteriol. 1995; 177: 1419-1424Crossref PubMed Google Scholar). There was no evidence of domain B in Rv3808c (and no conserved QXXRW amino acid motif) characteristic of β-glycosyltransferases that add sugars processively to the reducing end of a polysaccharide chain. β-Glycosyltransferases that add sugar residues to the nonreducing end of the polysaccharide chain have only the one domain, A (26Saxena I.M. Brown Jr., R.M. Fevre M. Geremia R.A. Henrissat B. J. Bacteriol. 1995; 177: 1419-1424Crossref PubMed Google Scholar). Thus, the synthesis of mycobacterial galactan may share similarities with biosynthesis of the homopolymer O-antigenic d-galactans of E. coliO8 and O9 and Klebsiella pneumoniae O1. In both cases, synthesis is initiated by the transfer of GlcNAc-P to polyprenyl-P (32Whitfield C. Trends Microbiol. 1995; 3: 178-185Abstract Full Text PDF PubMed Scopus (266) Google Scholar), and, during formation of the E. coli O9 antigen, mannosyl residues are rapidly added to the nonreducing terminus of the acceptor one residue at a time, in a processive mechanism (33Lind T. Lindhal U. Lindholt K. J. Biol. Chem. 1993; 268: 20705-20708Abstract Full Text PDF PubMed Google Scholar).d-Galactan I from K. pneumoniae is assembled on the cytoplasmic face of the plasma membrane, and polymerization is thought to occur by sequential sugar transfer on the lipid intermediate. An ATP-binding cassette (ABC) transporter then translocates polymerized D-galactan I across the plasma membrane prior to ligation to lipid A core (33Lind T. Lindhal U. Lindholt K. J. Biol. Chem. 1993; 268: 20705-20708Abstract Full Text PDF PubMed Google Scholar). The elucidation of the basic elements of synthesis of the cell wall core of mycobacteria should substantially enhance current tuberculosis drug discovery efforts, in that aspects of cell wall synthesis are the targets of many of the current front-line anti-tuberculosis drugs (2Crick D.C. Brennan P.J. Curr. Opin. Anti-Inf. Invest. Drugs. 2000; 2: 154-163Google Scholar), and the pathways and their end products are distinctly xenogeneic. We thank Caroline Morehouse for skilled technical assistance, Richard Lee for synthesis of decaprenyl-P-[1-14C]Araf, Michael Scherman for preparation of P[14C]ribosyl-PP, WenXin Yan for help in the preparation of dTDP-[14C]Rha, Yufang Ma for help in cloning Rv3808c, Carol Wassell for graphics, and Marilyn Hein for preparation of the manuscript." @default.
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W2031964267 title "Biosynthesis of the Galactan Component of the Mycobacterial Cell Wall" @default.
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