Construction of Escherichia Coli PldB Mutants to Investigate its Catalytic Mechanism
Katharine Sworden ’19 and Teresa Garrett (Chemistry)
PldB catalyzes the formation of PG, a head group acylated glycerophospholipid (HAGP) found in the Escherichia coli cell membrane that potentially impacts cell division. Little is known about the role of HAGP’s and research focusing on HAGP’s biosynthesis may reveal insights as to the biochemical role of these lipids in E. coli. PldB catalyzes the reaction of transferring an acyl chain from 2-acyl lyso PE to PG to form Acyl PG, but also functions as a lyso phospholipid lipase. To fully understand the mechanism of this reaction, variants of PldB were constructed based on its homology to alpha/beta hydrolases which contain a catalytic triad. Ser-139, Glu-270, and His-305 were individually mutated to alanine. Plasmids containing commercially synthesized genes of each mutation were obtained. Following transformation into DH5α cells, plasmids were purified and the PldB gene was isolated by restriction enzyme digestion followed by gel purification. Mutant PldB inserts were ligated into similarly digested pET 15b vector and then transformed into DH5α cells. Successful constructs were identified, sequenced, and transformed into C43 cells for protein expression. Cell Free extracts containing each mutant PldB have been prepared. Assays will be performed to characterize the affect these mutations have on the reaction versus the original PldB protein.