The ability of Gram-negative bacteria to modulate outer membrane (OM)
composition in response to stressful environments is essential for their survival
and replication within host tissues. The OM enzyme PagP catalyzes the transfer of
palmitate from a glycerophospholipid to lipid A. Lipid A is the endotoxic portion
of LPS responsible for transmembrane signalling to initiate the immune response.
Palmitoylation of lipid A can either attenuate or stimulate the immune response
depending on where the palmitate chain is attached to a specific lipid A molecule.
Here we report that the Escherichia coli PagP homolog is a multifunctional
enzyme, which displays two distinct active sites exposed on either side of the
bacterial OM. E. coli PagP converts phosphatidylglycerol (PG) to palmitoyl-PG
(PPG) using the same cell surface active site involved in the palmitoylation of
lipid A. PPG is then serially degraded to bis(monoacylglycero)phosphate (BMP)
and either lyso-PG or lyso-BMP by a novel lipase active site located in PagP on
the periplasmic side of the OM. The periplasmic lipase active site can be
inactivated with the Y87F amino acid substitution. BMP is a novel
glycerophosphoglycerol (GPG) that has not previously been reported in bacterial
lipid metabolism. Not all PagP homologs have this ability to remodel GPGs. We
have identified a divergent lipid A palmitoyltransferase in Pseudomonas
aeruginosa that does not palmitoylate PG. The P. aeruginosa homolog also has
different lipid A regiospecificity, adding palmitate on the opposite glucosamine at the 3’-position compared to the 2-position of the proximal sugar observed for the
E. coli homolog. We have determined that P. aeruginosa PagP is representative of
a distinct clade of PagP evolved to fulfill different functions. Although this minor
clade is inclusive of homologs that lack obvious sequence similarity with the
major clade enterobacterial PagP, we have identified conserved catalytic and
structural elements within the minor clade that contribute to our growing
understanding of homologous PagP structure/function relationships. A
comparative analysis of all available sequences of minor clade PagP homologs has
revealed invariant His, Ser, and Tyr residues that are necessary for catalysis.
Additionally, a 4-amino acid conserved signature indel or CSI is unique to
bacteria clustered phylogenetically within the γ-subclass of Proteobacteria. / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/24146 |
| Date | 22 November 2018 |
| Creators | Dixon, Charneal Latoye |
| Contributors | Bishop, Russell, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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