Lipopolysaccharide Core Glycosylation in Rhizobium leguminosarum

Abstract

The lipopolysaccharide structure of the nitrogen-fixing bacterium Rhizobium leguminosarum differs from that of Escherichia coli in several ways, one of which is the sugar composition of the core. The E. coli inner core consists of 3-deoxy-D-manno-octulosonic acid (Kdo) and L-glycero-D-manno-heptose (heptose), while the inner core of R. leguminosarum contains 2-keto-3-deoxy-Dmanno-octulosonic acid (Kdo), mannose, galactose, and galacturonic acid. The two Kdo residues and their linkages appear to be identical in both species. The linkages of heptose in E. coli and of mannose in R. leguminosarum to Kdo are both ␣1-5. We now characterize a membrane-associated glycosyl transferase in R. leguminosarum extracts that incorporates mannose into nascent lipopolysaccharide, using Kdo 2 -lipid IV A as the acceptor and GDP-mannose (or synthetic ADP-mannose) as the donor. The mannosyl transferase is associated with the inner membrane. The apparent K m values for GDP-mannose and Kdo 2 -lipid IV A are 4.3 M and 7.1 M, respectively, in the presence of excess co-substrate. Extracts of E. coli do not catalyze GDP-mannose-dependent glycosylation of Kdo 2 -lipid IV A , but they are active when ADPmannose is substituted for GDP-mannose. Given the structural similarity of ADP-mannose to ADP-heptose, we examined the possibility that heptosyl transferase I of E. coli (the product of the rfaC gene) catalyzes mannose transfer from ADP-mannose to Kdo 2 -lipid IV A . Extracts of E. coli mutants defective in the rfaC gene are unable carry out ADP-mannose-dependent glycosylation of Kdo 2 -lipid IV A . Plasmids bearing rfaC ؉ not only restore the missing activity but also direct its overexpression. Our assay using ADP-mannose as a substitute for ADP-heptose (which is not readily available) should facilitate the purification and characterization of heptosyl transferase I of E. coli. The GDP-mannose-dependent enzyme of R. leguminosarum may represent a functional equivalent of E. coli RfaC.

Cloning, expression and characterization of YSA1H, a human adenosine 5′-diphosphosugar pyrophosphatase possessing a MutT motif

“…However, ADP-sugars other than ADP-ribose are not significant metabolites in mammalian cells. ADP-glucose is an important precursor for bacterial glycogen and plant starch synthesis [27], whereas ADP-mannose has no known physiological function, although the commercially available synthetic compound can replace ADP-heptoses in bacterial outer-membrane lipopolysaccharide synthesis in itro [28]. ADP-ribose might therefore also be the most important substrate for human YSA1H in i o.…”

Cloning and Overexpression of Glycosyltransferases That Generate the Lipopolysaccharide Core of Rhizobium leguminosarum

“…Membranes of wild type R. leguminosarum strain 3841 were found to catalyze all three glycosylations (Fig. 5), using the assay conditions previously optimized for the mannosyltransferase (44). In these reactions, inclusion of GDPmannose alone was sufficient to cause a downward shift of the , but the CMP-Kdo-generating system by itself had no effect (lane 7).…”

“…In both systems, Kdo 2 -lipid IV A can be further acylated (2, 48 -50), but in R. leguminosarum, Kdo 2 -lipid IV Acan also be dephosphorylated at the 1-and 4Ј-positions to generate an unusual lipid A moiety lacking phosphate (23,43). In both systems, Kdo 2 -lipid IV A can also serve as an acceptor of several distinct core sugars, which are transferred one at a time from sugar nucleotide donors (18,19,23,44). A disadvantage of the E. coli core is the presence of L-glycero-D-mannoheptose (Fig.…”

“…A disadvantage of the E. coli core is the presence of L-glycero-D-mannoheptose (Fig. 1), the activated sugar nucleotide form of which is not fully characterized (2,18,19,44). Consequently, the enzymology of core glycosylation is more amenable to study in extracts of R. leguminosarum than of E. coli.…”

The Structures of the Lipopolysaccharides from Rhizobium etli Strains CE358 and CE359

“…Several of these enzyme activities have been reported, including a membranebound phosphatase that removes the 4Ј-phosphate from Kdo 2 lipid-IVa and requires the presence of the Kdo residues for maximum activity (21), a second phosphatase that removes the 1-phosphate (22), and a unique acyl carrier protein required for the transfer of the 27-hydroxyoctacosanoyl residue to the lipid A (23). In addition, a rhizobial-specific mannosyl transferase activity has been identified that transfers mannose from GDPMan to Kdo 2 lipid-IVa (24). It is apparent from these reports that R. leguminosarum and R. etli contain novel enzymes that can modify the enteric lipid A precursor, Kdo 2 lipid-IVa.…”