2.3.1.191 (3R)-3-hydroxyarachidonoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine the external layer of the Gram-negative bacterial outer membrane is primarily composed of a protective, selectively permeable lipopolysaccharide. The biosynthesis of lipopolysaccharide relies on UDP-3-O-acyl-glucosamine N-acyltransferase (LpxD), which transfers 3-hydroxy-arachidonic acid from acyl carrier protein to the 2' amine of UDP-3-O-myristoyl glucosamine. CtLpxD is expected to utilize R-3-hydroxyarachidonoyl-[acyl-carrier protein] and UDP-3-O-(myristoyl)-R-D-glucosamine, based on the predominant molecular species of Chlamydia trachomatis lipid A. This proposal is not validated by in vitro assays Chlamydia trachomatis UDP-2-N-((3R)-3-hydroxyarachidonoyl)-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein] - ? 402282 2.3.1.191 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine a comparison of the lipid A structures shows that in Escherichia coli and Neisseria meningitidis, LpxD can be expected to have the same specificity, both adding 3-hydroxymyristoyl chains Neisseria meningitidis UDP-2,3-bis((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein] - ? 402283 2.3.1.191 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine since only (R)-3-hydroxymyristate is found at the 2,3,2’, and 3’ positions of Escherichia coli lipid A, it is reassuring that both Escherichia coli acyltransferases display extraordinary specificity for (R)-3-hydroxymyristoyl-[acyl-carrier protein] Escherichia coli UDP-2,3-bis((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein] - ? 402283 2.3.1.191 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine myristoyl-[acyl-carrier protein] does not serve as an acyl donor for the overproduced UDP-3-O-((R)-3-hydroxymyristoyl)-GlcN N-acyltransferase Escherichia coli UDP-2,3-bis((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein] - ? 402283 2.3.1.191 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine third step of lipid A biosynthesis Escherichia coli UDP-2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein] - ? 402285 2.3.1.191 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine compulsory ordered mechanism in which (3R)-3-hydroxymyristoyl-[acyl-carrier protein] binds prior to UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine. The product, UDP-2,3-diacylglucosamine, dissociates prior to acyl-carrier protein Escherichia coli UDP-2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein] - ? 402285 2.3.1.191 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-[(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine - Escherichia coli UDP-2,3-bis[O-(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine + a holo-[acyl-carrier protein] - ir 419221 2.3.1.191 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-[(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine - Francisella tularensis subsp. novicida UDP-2,3-bis[O-(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine + a holo-[acyl-carrier protein] - ir 419221 2.3.1.191 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-[(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine - Arabidopsis thaliana UDP-2,3-bis[O-(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine + a holo-[acyl-carrier protein] - ir 419221 2.3.1.191 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-[(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine the LpxD1 enzyme adds a 3-OH C18 acyl group at 37 °C (host), whereas the LpxD2 enzyme adds a 3-OH C16 acyl group at 18 °C (environment) Francisella tularensis subsp. novicida UDP-2,3-bis[O-(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine + a holo-[acyl-carrier protein] - ir 419221 2.3.1.191 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-[(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine - Francisella tularensis subsp. novicida U112 UDP-2,3-bis[O-(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine + a holo-[acyl-carrier protein] - ir 419221 2.3.1.191 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-[(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine the LpxD1 enzyme adds a 3-OH C18 acyl group at 37 °C (host), whereas the LpxD2 enzyme adds a 3-OH C16 acyl group at 18 °C (environment) Francisella tularensis subsp. novicida U112 UDP-2,3-bis[O-(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine + a holo-[acyl-carrier protein] - ir 419221 2.3.1.191 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-[(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine - Arabidopsis thaliana Col-0 UDP-2,3-bis[O-(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine + a holo-[acyl-carrier protein] - ir 419221 2.3.1.191 (R,S)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine wild-type LpxD prefers (R,S)-3-hydroxymyristoyl-[acyl-carrier protein] over (R,S)-3-hydroxypalmitoyl-[acyl-carrier protein] by a factor of 3, whereas the M290A mutant has the opposite selectivity Escherichia coli UDP-2,3-bis((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein] - ? 403116 2.3.1.191 (R,S)-3-hydroxypalmitoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine wild-type LpxD prefers (R,S)-3-hydroxymyristoyl-[acyl-carrier protein] over (R,S)-3-hydroxypalmitoyl-[acyl-carrier protein] by a factor of 3, whereas the M290A mutant has the opposite selectivity Escherichia coli UDP-2,3-bis((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein] - ? 403117 2.3.1.191 additional information both LpxA and LpxD, from Escherichia coli are also able to incorporate odd-chain fatty acids into lipid A when grown in the presence of 1% propionic acid. When grown on 1% propionic acid lipid A also contains the odd-chain fatty acids tridecanoic acid (C13), pentadecanoic acid (C15), hydroxy tridecanoic acid (C13OH), and hydroxy pentadecanoic acid (C15OH). Escherichia coli lipid A acyltransferases do not have an absolute specificity for 14-carbon hydroxy fatty acids but can transfer fatty acids differing by one carbon unit if the fatty acid substrates are available Escherichia coli ? - ? 89 2.3.1.191 additional information His247 and His284 contribute to a mechanism involving nucleophilic attack by the amine of one substrate on the carbonyl carbon of an acyl carrier protein thioester conjugate Chlamydia trachomatis ? - ? 89 2.3.1.191 additional information lipid A from strains expressing either of the Porphyromonas gingivalis transferases contains 16-carbon hydroxy fatty acids in addition to the normal Escherichia coli 14-carbon hydroxy fatty acids, demonstrating that these acyltransferases display a relaxed acyl chain length specificity. Both LpxA and LpxD, from either Escherichia coli or Porphyromonas gingivalis are also able to incorporate odd-chain fatty acids into lipid A when grown in the presence of 1% propionic acid. The relaxed specificity of the Porphyromonas gingivalis lipid A acyltransferases and the substrate availability account for the lipid A structural clusters that differ by 14 mass units observed in Porphyromonas gingivalis lipopolysaccharide preparations Porphyromonas gingivalis ? - ? 89 2.3.1.191 additional information R-3-hydroxylauroyl-methylphosphopantetheine is a very poor substrate. The specific activity, measured at either 0.01 mM or 1 mM (3R)-3-hydroxylauroylmethylphosphopantetheine as the acyl donor, is more than 100fold lower than with 0.01 mM (3R)-3-hydroxymyristoyl-[acyl-carrier protein] Escherichia coli ? - ? 89 2.3.1.191 additional information fluorescent enzyme assay, method optimization, overview Escherichia coli ? - ? 89 2.3.1.191 additional information ordered-sequential reaction mechanism. Acyl-ACP binds first to free LpxD forming a binary complex. ACP associates with the ACP-recognition domain and the acyl-4'-phosphopantetheine group packs into the hydrophobic N-channel. UDP-acyl-GlcN binds next, which initiates acyl transfer. In the ternary product complex, the 4'-phosphopantetheine arm of hydrolysed-acyl-ACP completely encloses the reaction chamber, blocking UDP-diacyl-GlcN from leaving. By moving the 4'-phosphopantetheine group towards Met 290, the catalytic chamber opens up. This motion drives the eventual release of UDP-diacyl-GlcN and triggers conformational changes downstream of helix-II leading to holo-ACP dissociation Escherichia coli ? - ? 89