Biosynthesis of the Nod factor chito-oligosaccharide backbone in Rhizobium fredii is controlled by the concentration of UDP-N-acetyl-D-glucosamine.
1995
Inon de Iannino N. | Pueppke S.G. | Ugalde R.A.
A cell-free system was used to study the biochemical properties of the beta(1-4) N-acetyl-D-glucosaminyl transferase that forms the Nod factor chito-oligosaccharide backbone in Rhizobium fredii. Total membrane fractions prepared from a genistein-induced culture of wild-type R. fredii USDA257 incorporated N-acetyl-D-glucosamine at the rate of 6.0 nmoles.h-1.mg-1 protein into a beta(1-4) N-acetyl-D-glucosamine pentasaccharide. The Km for the substrate UDP-N-acetyl-D-glucosamine was 42 micromolar. For maximal activity, the reaction required free N-acetyl-D-glucosamine that could not be substituted by glucosamine, N-acetyl-D-galactosamine, or galactosamine. Chain elongation of the oligosaccharide proceeded toward the nonreducing end. Pulse-chase experiments demonstrated that the reaction follows a single chain mechanism; free chitobiose, chitotriose or chitotetraose were not substrates for elongation and/or initiation. The concentration of UDP-N-acetyl-D-glucosamine controlled the degree of polymerization of the chito-oligosaccharide formed in vitro. At low concentration (0.1 micromolar) the main product was chitobiose and chitotriose, and at concentrations higher than 10 micromolar, chitopentaose was the main product. Polyoxln D (200 micromolar) had no effect on the synthesis and or the degree of polymerization of the chito-oligosaccharide formed.
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