Dodecaprenyl phosphate-galacturonic acid as a donor substrate for lipopolysaccharide core glycosylation in rhizobium leguminosarum

doi:10.1074/jbc.M513865200

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Papers In Press, published online ahead of print February 23, 2006
J. Biol. Chem, 10.1074/jbc.M513865200
Submitted on December 29, 2005
Revised on February 22, 2006
Accepted on February 23, 2006

Dodecaprenyl phosphate-galacturonic acid as a donor substrate for lipopolysaccharide core glycosylation in rhizobium leguminosarum

Suparna Kanjilal-Kolar and Christian R. H. Raetz

Biochemistry, Duke University Medical Center, Durham, NC 27710

Corresponding Author: Raetz{at}biochem.duke.edu

The lipid A and inner core regions of Rhizobium leguminosarum lipopolysaccharide (LPS) contain four galacturonic acid (GalA) residues. Two are attached to the outer unit of the Kdo disaccharide, one to the mannose residue, and one to the 4'-position of lipid A. The enzymes RgtA and RgtB, described in the preceding manuscript, catalyze GalA transfer to the Kdo unit while RgtC is responsible for modification of the core mannose unit. Heterologous expression of RgtA in Sinorhizhobium meliloti 1021, a strain that normally lacks GalA modifications on its Kdo disaccharide, resulted in detectable GalA transferase activity in isolated membrane preparations, suggesting that the appropriate GalA donor substrate is available in S. meliloti membranes. In contrast, heterologous expression of RgtA in Escherichia coli yielded inactive membranes. However, RgtA activity was detectable in the E. coli system when total lipids from R. leguminosarum 3841 or S. meliloti 1021 were added. We have now purified and characterized dodecaprenyl (C60) phosphate-GalA as a minor novel lipid of R. leguminosarum 3841 and S. meliloti. This substance is stable to mild base hydrolysis and was purified by DEAE-cellulose column chromatography. Its structure was established by a combination of electrospray ionization mass spectrometry (ESI/MS) and gas-liquid chromatography (GC/MS). Purified dodecaprenyl phosphate-GalA supports the efficient transfer of GalA to Kdo2-1-dephospho-lipid IVA by membranes of E. coli cells expressing RgtA, RgtB and RgtC. The identification of a polyisoprene phosphate-GalA donor substrate suggests that the active site of RgtA faces the periplasmic side of the inner membrane. This work represents the first definitive characterization of a lipid-linked GalA derivative with the proposed structure dodecaprenyl phosphate-ß-D-GalA.

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