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The synthesis of exopolysaccharide by Klebsiella aerogenes membrane preparations and the involvement of lipid intermediates.

ABSTRACT: 1. Membrane preparations from Klebsiella aerogenes type 8 were shown to transfer glucose and galactose from their uridine diphosphate derivatives to a lipid and to polymer. The ratio of glucose to galactose transfer in both cases was 1:2. This is the same ratio in which these sugars occur in native polysaccharide. Galactose transfer was dependent on prior glucosylation of the lipid. Mutants were obtained lacking (a) glucosyltransferase and (b) galactosyltransferase. The transferase activities in a number of non-mucoid mutants was examined. 2. Glucose transfer was partially inhibited by uridine monophosphate, and incorporation of either glucose or galactose into lipid was decreased in the presence of uridine diphosphate. The sugars are thought to be linked to a lipid through a pyrophosphate bond, and treatment of the lipid intermediates with phenol yielded water-soluble compounds. These could be dephosphorylated with alkaline phosphatase. Transfer of glucuronic acid to lipid or polymer from uridine diphosphate glucuronic acid was much lower than that of the other two sugars. 3. The fate of sugars incorporated into polymer was also followed. Some conversion of glucose into galactose and glucuronic acid occurred. Mutants unable to transfer glucose or galactose to lipid were unable to form polymer. Other mutants capable of lipid glycosylation were in some cases unable to form polymer. A model for capsular polysaccharide synthesis is proposed and its similarity to the formation of other polymers outside the cell membrane is discussed.

SUBMITTER: Sutherland IW

PROVIDER: S-EPMC1179638 | biostudies-other | 1970 Dec

REPOSITORIES: biostudies-other

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Project description:Urease from Klebsiella aerogenes is composed of three subunits (UreA-UreB-UreC) that assemble into a (UreABC)(3) quaternary structure. UreC harbors the dinuclear nickel active site, whereas the functions of UreA and UreB remain unknown. UreD and UreF accessory proteins previously were suggested to reposition UreB and increase the level of exposure of the nascent urease active site, thus facilitating metallocenter assembly. In this study, cells were engineered to separately produce (UreAC)(3) or UreB, and the purified proteins were characterized. Monomeric UreB spontaneously binds to the trimeric heterodimer of UreA and UreC to form (UreABC*)(3) apoprotein, as shown by gel filtration chromatography, integration of electrophoretic gel band intensities, and mass spectrometry. Similar to the authentic urease apoprotein, the active enzyme is produced by incubation of (UreABC*)(3) with Ni(2+) and bicarbonate. Conversely, UreB?1-19, lacking the 19-residue potential hinge and tether to UreC, does not form a complex with (UreAC)(3) and yields negligible levels of the active enzyme when incubated under activation conditions with (UreAC)(3). Comparison of activities and nickel contents for (UreAC)(3), (UreABC*)(3), and (UreABC)(3) samples treated with Ni(2+) and bicarbonate and then desalted indicates that UreB facilitates efficient incorporation of the metal into the active site and protects the bound metal from chelation. Amylose resin pull-down studies reveal that MBP-UreD (a fusion of maltose binding protein with UreD) forms complexes with (UreABC)(3), (UreAC)(3), and UreB in vivo, but not in vitro. By contrast, MBP-UreD does not form an in vivo complex with UreB?1-19. The soluble MBP-UreD-UreF-UreG complex binds in vitro to (UreABC)(3), but not to (UreAC)(3) or UreB. Together, these data demonstrate that UreB facilitates the interaction of urease with accessory proteins during metallocenter assembly, with the N-terminal hinge and tether region being specifically required for this process. In addition to its role in urease activation, UreB enhances the stability of UreC against proteolytic cleavage.

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The synthesis of exopolysaccharide by Klebsiella aerogenes membrane preparations and the involvement of lipid intermediates.

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