Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris.
Lipid-linked intermediates are involved in the synthesis of the exopolysaccharide xanthan produced by the bacterium Xanthomonas campestris (L. Ielpi, R. O. Couso, and M. A. Dankert, FEBS Lett. 130:253-256, 1981). In this study, the stepwise assembly of the repeating pentasaccharide unit of xanthan is described. EDTA-treated X. campestris cells were used as both enzyme preparation and lipid-P acceptor, and UDP-Glc, GDP-Man, and UDP-glucuronic acid were used as sugar donors. A linear pentasaccharide unit is assembled on a polyprenol-P lipid carrier by the sequential addition of glucose-1-P, glucose, mannose, glucuronic acid, and mannose. The in vitro synthesis of pentasaccharide-P-P-polyprenol was also accompanied by the incorporation of radioactivity into a polymeric product, which was characterized as xanthan, on the basis of gel filtration and permethylation studies. Results from two-stage reactions showed that essentially pentasaccharide-P-P-polyprenol is polymerized. In addition, the direction of chain elongation has been studied by in vivo experiments. The polymerization of lipid-linked repeat units occurs by the successive transfer of the growing chain to a new pentasaccharide-P-P-polyprenol. The reaction involves C-1 of glucose at the reducing end of the polyprenol-linked growing chain and C-4 of glucose at the nonreducing position of the newly formed polyprenol-linked pentasaccharide, generating a branched polymer with a trisaccharide side chain.
Ielpi
L
L
Instituto de Investigaciones Bioquímicas Fundación Campomar, Facultad de Ciencias Exactas y Naturales, Buenos Aires, Argentina.
Couso
R O
RO
Dankert
M A
MA
D002240:Carbohydrate Sequence; D005947:Glucose / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000378:metabolism*; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism*; D011108:Polymers; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism; D016959:Xanthomonas campestris / Q000378:metabolism*
D005958:Glucosephosphates; D011103:Polyisoprenyl Phosphate Monosaccharides; D011104:Polyisoprenyl Phosphate Oligosaccharides; D011108:Polymers; D011135:Polysaccharides, Bacterial; D014535:Uridine Diphosphate Glucuronic Acid; D006155:Guanosine Diphosphate Mannose; C031590:glucose-1-phosphate; D005947:Glucose; D008358:Mannose; C002563:xanthan gum; D014532:Uridine Diphosphate Glucose