A Chlamydia trachomatis UDP-N-acetylglucosamine acyltransferase selective for myristoyl-acyl carrier protein: Expression in Escherichia coli and formation of hybrid lipid A species

Charles R. Sweet, Shanhua Lin, Robert J. Cotter, Christian R H Raetz

Research output: Contribution to journalArticle

Abstract

Chlamydia trachomatis lipid A is unusual in that it is acylated with myristoyl chains at the glucosamine 3 and 3′ positions. We have cloned and expressed the gene encoding UDP-N. acetylglucosamine 3-O-acyltransferase of C. trachomatis (CtlpxA), the first enzyme of lipid A biosynthesis. C. trachomatis LpxA displays ∼20-fold selectivity for myristoyl-ACP over R/S-3-hydroxymyristoyl-ACP under standard assay conditions, consistent with the proposed structure of C. trachomatis lipid A. CtLpxA is the first reported UDP-N-acetylglucosamine acyltransferase that prefers a non-hydroxylated acyl-ACP to a hydroxyacyl-ACP. When CtlpxA was expressed in R0138, a temperature-sensitive lpxA mutant of Escherichia coli, five new hybrid lipid A species were made in vivo after 2 h at 42 °C, in place of Escherichia coli lipid A. These compounds were purified and analyzed by matrix-assisted laser desorption ionization/time of flight mass spectrometry. In each case, a myristoyl chain replaced one or both of the ester linked 3-hydroxymyristoyl residues of E. coli lipid A. With prolonged growth at 42 °C, all the ester-linked 3-hydroxymyristoyl residues were replaced with myristate chains. Re-engineering the structure of E. coli lipid A should facilitate the microbiological production of novel agonists or antagonists of the innate immunity receptor TLR-4, with possible uses as adjuvants or anti-inflammatory agents.

Original languageEnglish (US)
Pages (from-to)19565-19574
Number of pages10
JournalJournal of Biological Chemistry
Volume276
Issue number22
DOIs
StatePublished - Jun 1 2001

Fingerprint

Uridine Diphosphate N-Acetylglucosamine
Acyl Carrier Protein
Acyltransferases
Lipid A
Uridine Diphosphate
Acetylglucosamine
Chlamydia trachomatis
Escherichia coli
Esters
Gene encoding
Glucosamine
Biosynthesis
Myristic Acid
Innate Immunity
Ionization
Mass spectrometry
Assays
Desorption
Mass Spectrometry
Lasers

ASJC Scopus subject areas

  • Biochemistry

Cite this

A Chlamydia trachomatis UDP-N-acetylglucosamine acyltransferase selective for myristoyl-acyl carrier protein : Expression in Escherichia coli and formation of hybrid lipid A species. / Sweet, Charles R.; Lin, Shanhua; Cotter, Robert J.; Raetz, Christian R H.

In: Journal of Biological Chemistry, Vol. 276, No. 22, 01.06.2001, p. 19565-19574.

Research output: Contribution to journalArticle

@article{f97b46a4093747d085a851c96d8a48b3,
title = "A Chlamydia trachomatis UDP-N-acetylglucosamine acyltransferase selective for myristoyl-acyl carrier protein: Expression in Escherichia coli and formation of hybrid lipid A species",
abstract = "Chlamydia trachomatis lipid A is unusual in that it is acylated with myristoyl chains at the glucosamine 3 and 3′ positions. We have cloned and expressed the gene encoding UDP-N. acetylglucosamine 3-O-acyltransferase of C. trachomatis (CtlpxA), the first enzyme of lipid A biosynthesis. C. trachomatis LpxA displays ∼20-fold selectivity for myristoyl-ACP over R/S-3-hydroxymyristoyl-ACP under standard assay conditions, consistent with the proposed structure of C. trachomatis lipid A. CtLpxA is the first reported UDP-N-acetylglucosamine acyltransferase that prefers a non-hydroxylated acyl-ACP to a hydroxyacyl-ACP. When CtlpxA was expressed in R0138, a temperature-sensitive lpxA mutant of Escherichia coli, five new hybrid lipid A species were made in vivo after 2 h at 42 °C, in place of Escherichia coli lipid A. These compounds were purified and analyzed by matrix-assisted laser desorption ionization/time of flight mass spectrometry. In each case, a myristoyl chain replaced one or both of the ester linked 3-hydroxymyristoyl residues of E. coli lipid A. With prolonged growth at 42 °C, all the ester-linked 3-hydroxymyristoyl residues were replaced with myristate chains. Re-engineering the structure of E. coli lipid A should facilitate the microbiological production of novel agonists or antagonists of the innate immunity receptor TLR-4, with possible uses as adjuvants or anti-inflammatory agents.",
author = "Sweet, {Charles R.} and Shanhua Lin and Cotter, {Robert J.} and Raetz, {Christian R H}",
year = "2001",
month = "6",
day = "1",
doi = "10.1074/jbc.M101868200",
language = "English (US)",
volume = "276",
pages = "19565--19574",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "22",

}

TY - JOUR

T1 - A Chlamydia trachomatis UDP-N-acetylglucosamine acyltransferase selective for myristoyl-acyl carrier protein

T2 - Expression in Escherichia coli and formation of hybrid lipid A species

AU - Sweet, Charles R.

AU - Lin, Shanhua

AU - Cotter, Robert J.

AU - Raetz, Christian R H

PY - 2001/6/1

Y1 - 2001/6/1

N2 - Chlamydia trachomatis lipid A is unusual in that it is acylated with myristoyl chains at the glucosamine 3 and 3′ positions. We have cloned and expressed the gene encoding UDP-N. acetylglucosamine 3-O-acyltransferase of C. trachomatis (CtlpxA), the first enzyme of lipid A biosynthesis. C. trachomatis LpxA displays ∼20-fold selectivity for myristoyl-ACP over R/S-3-hydroxymyristoyl-ACP under standard assay conditions, consistent with the proposed structure of C. trachomatis lipid A. CtLpxA is the first reported UDP-N-acetylglucosamine acyltransferase that prefers a non-hydroxylated acyl-ACP to a hydroxyacyl-ACP. When CtlpxA was expressed in R0138, a temperature-sensitive lpxA mutant of Escherichia coli, five new hybrid lipid A species were made in vivo after 2 h at 42 °C, in place of Escherichia coli lipid A. These compounds were purified and analyzed by matrix-assisted laser desorption ionization/time of flight mass spectrometry. In each case, a myristoyl chain replaced one or both of the ester linked 3-hydroxymyristoyl residues of E. coli lipid A. With prolonged growth at 42 °C, all the ester-linked 3-hydroxymyristoyl residues were replaced with myristate chains. Re-engineering the structure of E. coli lipid A should facilitate the microbiological production of novel agonists or antagonists of the innate immunity receptor TLR-4, with possible uses as adjuvants or anti-inflammatory agents.

AB - Chlamydia trachomatis lipid A is unusual in that it is acylated with myristoyl chains at the glucosamine 3 and 3′ positions. We have cloned and expressed the gene encoding UDP-N. acetylglucosamine 3-O-acyltransferase of C. trachomatis (CtlpxA), the first enzyme of lipid A biosynthesis. C. trachomatis LpxA displays ∼20-fold selectivity for myristoyl-ACP over R/S-3-hydroxymyristoyl-ACP under standard assay conditions, consistent with the proposed structure of C. trachomatis lipid A. CtLpxA is the first reported UDP-N-acetylglucosamine acyltransferase that prefers a non-hydroxylated acyl-ACP to a hydroxyacyl-ACP. When CtlpxA was expressed in R0138, a temperature-sensitive lpxA mutant of Escherichia coli, five new hybrid lipid A species were made in vivo after 2 h at 42 °C, in place of Escherichia coli lipid A. These compounds were purified and analyzed by matrix-assisted laser desorption ionization/time of flight mass spectrometry. In each case, a myristoyl chain replaced one or both of the ester linked 3-hydroxymyristoyl residues of E. coli lipid A. With prolonged growth at 42 °C, all the ester-linked 3-hydroxymyristoyl residues were replaced with myristate chains. Re-engineering the structure of E. coli lipid A should facilitate the microbiological production of novel agonists or antagonists of the innate immunity receptor TLR-4, with possible uses as adjuvants or anti-inflammatory agents.

UR - http://www.scopus.com/inward/record.url?scp=0035378670&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035378670&partnerID=8YFLogxK

U2 - 10.1074/jbc.M101868200

DO - 10.1074/jbc.M101868200

M3 - Article

C2 - 11279221

AN - SCOPUS:0035378670

VL - 276

SP - 19565

EP - 19574

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 22

ER -