Biosynthesis of N-acetylmannosaminuronic-acid-containing cell-wall polysaccharide of Bacillus subtilis.
The particulate enzyme from Bacillus subtilis AHU 1031 catalyzed the synthesis of a polysaccharide and glycolipids from UDP-N-acetylmannosaminuronic acid (UDP-ManNAcUA), UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-glucose (UDP-Glc). The polysaccharide synthesis required UDP-ManNAcUA and UDP-GlcNAc, proceeded optimally at pH 8.5 and in the presence of 5 mM MgCl2 and 2.5 mM dithiothreitol, and was stimulated by the addition of UDP-Glc. The molar ratio of ManNAcUA, GlcNAc, and Glc incorporated into polysaccharide was calculated to be 1:1:1.8 from chemical analysis involving reduction with water soluble carbodiimide; its relative molecular mass was estimated to be 12000. The analysis of Smith degradation products revealed that the polysaccharide backbone is composed of repeating trisaccharide units comprising ManNAcUA, GlcNAc, and Glc. Based on the data regarding the time course of the incorporation of glucose into the polysaccharide, extra glucose seems to be attached to the polysaccharide backbone as lateral branches. The saccharide moieties of the glycolipids were identified as GlcNAc, ManNAcUA-GlcNAc, and Glc-ManNAcUA-GlcNAc from several analytical criteria. The addition of antibiotic 24010, a tunicamycin-like antibiotic, at 10 micrograms/ml resulted in almost complete inhibition of the synthesis of glycolipids and polysaccharide. It is therefore concluded that the glycolipids function as intermediates in polysaccharide formation. Incubation of the ManNAcUA-GlcNAc-linked lipid. (labeled in the ManNAcUA moiety) with the particulate enzyme and UDP-Glc resulted incorporation of radioactivity into a trisaccharide-linked lipid and a polysaccharide. These results suggest that the particulate enzyme utilizes the trisaccharide moiety of the Glc-ManNAcUA-GlcNAc-linked lipid for the elongation of the main polysaccharide chain presumed to be cell wall acidic polysaccharide of this strain.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001412:Bacillus subtilis / Q000201:enzymology*; D002384:Catalysis; D002473:Cell Wall / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D006017:Glycolipids / Q000096:biosynthesis; D008670:Metals / Q000494:pharmacology; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014415:Tunicamycin / Q000031:analogs & derivatives; D014530:Uridine Diphosphate / Q000494:pharmacology; D014542:Uridine Monophosphate / Q000494:pharmacology; D014574:Uronic Acids / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D006017:Glycolipids; D008670:Metals; D011135:Polysaccharides, Bacterial; D013439:Sulfhydryl Reagents; D014574:Uronic Acids; D014415:Tunicamycin; C017012:antibiotic 24010; C043028:N-acetylmannosaminuronic acid; D014530:Uridine Diphosphate; D014542:Uridine Monophosphate