| The acylation of UDP-N-acetylglucosamine is the first step in the canonical biosynthesis of lipid A, a unique glycolipid that is the predominant structural component of the outer leaflet of the outer membrane of Gram-negative bacteria. This acylation reaction is catalyzed by UDP-N-acetylglucosamine acyltransferase, LpxA. The unusual substrates and substrate specificities of the LpxA proteins studied in this thesis generate distinct lipid A structural variations, as compared to the archetype structure of Escherichia coli, hexa-acyl Kdo 2-lipid A. This structural diversity may have important effects on the function of lipid A as a structural component of the cellular envelope and as endotoxin, a primary mediator of the eukaryotic innate immune response to Gram-negative infection. This dissertation describes elucidation of the genetic and enzymatic basis of these structural variations.; Specifically, this work explores (1) The presence of 3 and 3 ' primary acyl chains that lack hydroxyl groups in Chlamydia trachomatis, (2) The incorporation of multiple lengths of 3-OH acyl chain in 3 and 3' primary acyl positions of Bordetella species, and (3) The biosynthetic origin of lipid A containing 2,3-diamino-2,3-dideoxy-D-glucose as a backbone sugar. The latter effort involved not only the characterization of LpxA proteins with novel sugar nucleotide specificity, but also the discovery of two Acidithiobacillus ferrooxidans proteins, GnnA and GnnB, which are necessary and sufficient for the conversion of UDP-N-acetylglucosamine to UDP-2-N-acetyl-2,3-diamino-2,3-dideoxy-D-glucose, a novel sugar nucleotide that is the precursor of lipid A containing 2,3-diamino-2,3-dideoxy-D-glucose as a backbone sugar.; The substrate specificities of the LpxA acyltransferase enzymes were characterized by in vitro thin-layer chromatography assays of acyltransferase activity and in vivo by complementation of a temperature-sensitive E. coli mutant, lpxA2 . These activities were further examined by MALDI-TOF mass spectrometry of the hybrid lipid A structures isolated from the lpxA2 conditional mutant covered with the foreign lpxA genes. The A. ferrooxidans genes gnnA and gnnB were cloned and expressed in E. coli, and the proteins were purified and characterized by in vitro thin-layer chromatography activity assays. Furthermore, the in vitro reaction product UDP-2-N-acetyl-2,3-diamino-2,3-dideoxy-D-glucose was purified by ion-exchange chromatography and characterized by one- and two-dimensional NMR spectroscopy. |
