A Chlamydia trachomatisUDP-N-Acetylglucosamine Acyltransferase Selective for Myristoyl-Acyl Carrier Protein

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 ϳ20-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°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.

Structural basis for the acyl chain selectivity and mechanism of UDP- N -acetylglucosamine acyltransferase

“…The in vitro fatty acyl chain length selectivity of these LpxA orthologues is consistent with the structures of the lipid A molecules isolated from E. coli and P. aeruginosa (2). The only known LpxA variant that does not require R-3-hydroxyacyl-ACP is that of Chlamydia trachomatis, which shows a strong preference for myristoyl-ACP over R-3-hydroxymyristoyl-ACP (21). E. coli LpxA is inhibited by myristoyl-ACP (8).…”

“…4). Interestingly, H99 is replaced by threonine in C. trachomatis LpxA (21). This substitution may account for loss of R-3-hydroxyacyl chain selectivity, because threonine cannot replace histidine as a hydrogen-bonding partner.…”

Enzymatic Synthesis of Lipid A Molecules with Four Amide-linked Acyl Chains

“…The acyl-ACP donor selectivity of LpxA has previously been studied in several systems (8,17,24,(35)(36)(37). In general, LpxA orthologs show strong preferences for acyl chain length and the presence of the R-3-hydroxyl group (8,17,24,(35)(36)(37).…”

“…In general, LpxA orthologs show strong preferences for acyl chain length and the presence of the R-3-hydroxyl group (8,17,24,(35)(36)(37). The corresponding coenzyme A thioesters are not substrates (8,25).…”

“…Furthermore, LiLpxA does not acylate UDP-GlcNAc3N with any donor other than 3-hydroxylauroyl-ACP. Although most other LpxA proteins display a high degree of specificity for a particular acyl chain length, they do in fact function with alternative acyl donors at slow rates (Tables II and III) (8,17,24,35,37).…”