Fluorescent Imaging Studies of Escherichia coli cis-type Undecaprenyl Pyrophosphate Synthase Interacting with Model Membrane
Date Issued
2008
Date
2008
Author(s)
Teng, Kuo-Hsun
Abstract
Undecaprenyl pyrophosphate synthase (UPPs) catalyzes eight consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C55-Undecaprenyl pyrophosphate (UPP), as a lipid carrier to mediate the synthesis of bacterial cell wall peptidoglycans. UPPs is highly soluble when overexpressed in Escherichia coli, but its product, UPP, is membrane bound. It is unknown how the protein transfers its product to bacterial cell membrane. In order to study the mechanism in which how UPPs adhered to bacterial cell membrane, we employed fluorescent imaging technology to observe the dynamics of UPPs in different cell membranes in the absence and presence of different ligands. From the results, with or without substrates, UPPs always adhered to the membrane mixed with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and 1, 2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG) in the ratio of 1:3, which mimicks E. coli. membrane with both negative charges and hydrogen bond-forming groups in it. The net charge in the membrane determines the adhesion of UPPs. On the other hand, during the synthesis of UPP, UPPs adhered to 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membrane, which had neither net charge nor the polar group which can form hydrogen bonds in it. When the synthesis of UPP was blocked, such as the addition of farnesyl s-thiolodiphosphate (FsPP), which was FPP-analog to inhibit UPPs to synthesize UPP, the elimination of Mg2+, UPPs did not adhere to DPPC membrane. Mutant D26A-UPPs partially adhered to the DPPC membrane in the presence of FPP and IPP, indicating that during the synthesis of UPP, UPPs gradually moves from the defect regions to membrane. However, mutant S83(Ala)5-UPPs, which fixed UPPs in the open form, adhered to DPPC membrane in the absence and presence of the substrates, even though its activity is 103~104 fold lower compared to the wild-type UPPs. In addition, in the presence of IPP, UPPs adhered to DPPC membrane. The elimination of Mg2+ and mutant form of D26A caused UPPs not to adhere to membrane. It seems that IPP binding plays an important role on the adhesion of UPPs to the membrane. Therefore, the net charge of the membrane, the conformational change of UPPs during the synthesis of UPP, and IPP binding determine whether UPPs can adhere to the bacterial cell membrane.
Subjects
Undecaprenyl Pyrophosphate Synthase
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