Abstract
The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo2-lipid IVA. LpcC containing an N-terminal His6 tag was assayed using GDP-mannose as the donor and Kdo2-[4′-32P]lipid IVA as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcC gene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo2-[4′-32P]lipid IVA. A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IVA or Kdo-lipid IVA. The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the α-anomeric configuration) for the glycosylation of Kdo2-[4′-32P]lipid IVA. Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the inner L-glycero-D-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented with lpcC. An Escherichia coli heptose-less waaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo2-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella and E. coli LPS cores in living cells.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 16356-16364 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 278 |
| Issue number | 18 |
| DOIs | |
| State | Published - May 2 2003 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum : Purification, substrate specificity, and expression in Salmonella waaC mutants. / Kanipes, Margaret I.; Ribeiro, Anthony A.; Lin, Shanhua; Cotter, Robert J.; Raetz, Christian R H.
In: Journal of Biological Chemistry, Vol. 278, No. 18, 02.05.2003, p. 16356-16364.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum
T2 - Purification, substrate specificity, and expression in Salmonella waaC mutants
AU - Kanipes, Margaret I.
AU - Ribeiro, Anthony A.
AU - Lin, Shanhua
AU - Cotter, Robert J.
AU - Raetz, Christian R H
PY - 2003/5/2
Y1 - 2003/5/2
N2 - The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo2-lipid IVA. LpcC containing an N-terminal His6 tag was assayed using GDP-mannose as the donor and Kdo2-[4′-32P]lipid IVA as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcC gene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo2-[4′-32P]lipid IVA. A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IVA or Kdo-lipid IVA. The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the α-anomeric configuration) for the glycosylation of Kdo2-[4′-32P]lipid IVA. Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the inner L-glycero-D-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented with lpcC. An Escherichia coli heptose-less waaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo2-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella and E. coli LPS cores in living cells.
AB - The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo2-lipid IVA. LpcC containing an N-terminal His6 tag was assayed using GDP-mannose as the donor and Kdo2-[4′-32P]lipid IVA as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcC gene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo2-[4′-32P]lipid IVA. A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IVA or Kdo-lipid IVA. The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the α-anomeric configuration) for the glycosylation of Kdo2-[4′-32P]lipid IVA. Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the inner L-glycero-D-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented with lpcC. An Escherichia coli heptose-less waaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo2-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella and E. coli LPS cores in living cells.
UR - http://www.scopus.com/inward/record.url?scp=0037600773&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037600773&partnerID=8YFLogxK
U2 - 10.1074/jbc.M301255200
DO - 10.1074/jbc.M301255200
M3 - Article
C2 - 12591937
AN - SCOPUS:0037600773
VL - 278
SP - 16356
EP - 16364
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 18
ER -