Core sugars

The biosynthesis in yeast of two enzymes that are D-mannoproteins has been studied. A membrane-associated isozyme of invertase (EC 3.2.1.26) has been shown to be a precursor of the external invertase.190 Its molecular weight, as determined by SDS-poly(acrylamide) gel electrophoresis, is 50,000, that is, smaller than that of the external invertase, and it correlates well with the presence of only the inner-core sugars of the final form. It is strictly bound to membranes, possibly those of the endoplasmic reticulum, and it can be completely split191 by endo-/3-N-acetylglucosaminidase H (EC 3.2.1.30). The addition of tunicamycin, which specifically inhibits formation of d-GIcNAc-PP-DoI, inhibits synthesis of external invertase, as well as further formation of the membrane-associated form, which completely disappears after addition of the antibiotic.190 In these aspects, the synthesis of this extracellular enzyme follows the pathway for secreted glycoproteins in animal systems. 

Reynolds, C.M., Raetz, C.R. Replacement of lipopolysaccharide with free lipid A molecules in Escherichia coli mutants lacking all core sugars. Biochemistry 48 (2009) 9627-9640. 

The lowest Mr fraction, OS-3, was in fact a mixture of core-related linear molecules (Fig. 6) containing a minimal disaccharide composed of the innermost LD-Hep (phosphorylated) and its neighboring Kdo residue, and three more extended glycans that allowed for the determination of the core OS backbone. The backbone of the core OS of H. pylori type strain was composed of a phosphorylated (monoester phosphate) hexasaccharide in which the typical core sugars, LD-Hep, Glc, and Gal, were present, but, in addition, this core OS also possessed the less common DDHep as a constituent. 

Fig. 7. Biosynthesis of endotoxin in E. coli. The first step (1) in the pathway is catalyzed by UDP-A -acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA). (2) The committed step is catalyzed by the LpxC deacetylase, followed by (3) a second acyltransferase (LpxD). (4) Lipid X is generated by the removal of UMP from UDP-2,3-diacyl-GlcN by an unknown enzyme. (5) Lipid X and UDP-2,3-diacyl-GlcN are then condensed together by LpxD to form Lipid IV. (6) A 4 -kinase phos-phorylates the disaccharide to produce lipid IV. (7) Two consecutive additions of KDO by KdtA, and two 0-acylations by (8) HtrB and (9) MsbB yield KDOj-lipid A. Subsequent addition of core sugars and 0-antigen chains (not shown) yield the mature LPS.

Lipid A. The ketosidic linkage between the terminal core sugar 3-deoxy-D-mfl/mo-octulosonic acid (KDO) and lipid A is acid labile. Hence the hydrophobic lipid A region can be easily separated from isolated LPS by mild acid hydrolysis, followed by chloroform extraction. The structural basis of the LPS extracted from enteric bacteria is a glucosamine disacch-... 

The lipopolysaccharide molecule can be divided into three distinct domains the lipid A region, the core sugars, and the O-antigen repeat. Figure IB shows a schematic representation of the LPS molecule. 

The core plays an important role in maintaining an effective permeability barrier. E. coli mutants lacking heptose and distal core sugars do not correctly insert all of the outer membrane porins and are more susceptible to antibiotics [7]. In other Gram-negative bacteria, such as Haemophilus influenzae, the LPS core is hyper variable and important in the organism s ability to evade immune detection [8, 9], In Rhizobium, disruption of core biosynthesis inhibits bacteriod formation in plant root nodules [10]. 

As noted above the entire LPS molecule is not required for growth of E. coli. The minimal portion that promotes growth under laboratory conditions is lipid A with at least one of its acyloxyacyl moieties (i.e. a penta-acylated species), and two Kdo sugars (see Figures 1 and 2). However, the core sugars and the O-antigen plays a role in virulence and other specialized functions, such as symbiosis in Rhizobia [4, 8, 10]. 

The enzymes required for the biosynthesis of Kdo2-lipid A and the addition of the core sugars are localized to the cytoplasm or the inner membrane of the cell [1], All of the substrates (e.g. acyl-ACP, sugar nucleotides) are also found in the cytoplasm... 

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