A Chlamydia trachomatis UDP-N-acetylglucosamine acyltransferase selective for myristoyl-acyl carrier protein. Expression in Escherichia coli and formation of hybrid lipid A species.
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 approximately 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 RO138, a temperature-sensitive lpxA mutant of Escherichia coli, five new hybrid lipid A species were made in vivo after 2 h at 42 degrees 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 degrees 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.
Sweet
C R
CR
Department of Biochemistry, Duke University, Durham, North Carolina 27710, USA.
Lin
S
S
Cotter
R J
RJ
Raetz
C R
CR
D000213:Acyl Carrier Protein / Q000737:chemistry*; D000217:Acyltransferases / Q000096:biosynthesis* / Q000737:chemistry*; D002455:Cell Division; D002692:Chlamydia trachomatis / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D008050:Lipid A / Q000096:biosynthesis*; D050356:Lipid Metabolism; D008956:Models, Chemical; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D000213:Acyl Carrier Protein; D008050:Lipid A; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000217:Acyltransferases; C050304:acyl-(acyl-carrier-protein)-UDP-N-acetylglucosamine acyltransferase