Dodecaprenyl Phosphate-Galacturonic Acid as a Donor Substrate for Lipopolysaccharide Core Glycosylation in Rhizobium leguminosarum

Suparna Kanjilal-Kolar,Christian R.H Raetz

doi:10.1074/jbc.m513865200

Abstract

The lipid A and inner core regions of Rhizobium leguminosarum lipopolysaccharide contain four galacturonic acid (GalA) residues. Two are attached to the outer unit of the 3-deoxy-D-manno-octulosonic acid (Kdo) disaccharide, one to the mannose residue, and one to the 4'-position of lipid A. The enzymes RgtA and RgtB, described in the accompanying article, catalyze GalA transfer to the Kdo residue, whereas 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 and gas-liquid chromatography. Purified dodecaprenyl phosphate-GalA supports the efficient transfer of GalA to Kdo2-1-dephospho-lipid IV(A) 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-beta-D-GalA.

Highlights

As shown in the preceding article [17], the membrane-bound enzymes RgtA, RgtB, and RgtC are responsible for the transfer of three galacturonic acid (GalA) moieties to the Rhizobium leguminosarum LPS2 core (Fig. 1)
Based on the sequence similarity of RgtA to better characterized enzymes that utilize dolichyl phosphate-mannose as donor substrates [12, 15], we suggest that the GalA unit of our dodecaprenyl phosphateGalA has the ␤-anomeric configuration
Based on the characteristics of the Rgt proteins and their sequence similarity to the E. coli ArnT [20, 21, 30], we hypothesized that a polyisoprene phosphate-linked GalA donor might be involved

Summary

Introduction

As shown in the preceding article [17], the membrane-bound enzymes RgtA, RgtB, and RgtC are responsible for the transfer of three GalA moieties to the Rhizobium leguminosarum LPS2 core (Fig. 1). The presence of a polyisoprene phosphate-linked GalA donor lipid suggests that GalA addition to the LPS core occurs on the periplasmic leaflet of the inner membrane.

Results

Conclusion