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Structural and functional characterization of enzymes central to bacterial carrier lipid synthesis and recycling

University of British Columbia

In bacteria, the carrier lipid undecaprenyl phosphate (C₅₅P) is used as a scaffold for the synthesis of bacterial cell wall polymers such as peptidoglycan. C55P is synthesized as undecaprenyl pyrophosphate (C55PP) by undecaprenyl pyrophosphate synthase (UppS) and must be dephosphorylated by an as yet unknown mechanism before it can be used in cell wall biosynthesis. Individual subunits of cell wall polymers are assembled in the cytoplasm on C₅₅P before being flipped to the periplasmic face of the membrane where they are polymerized into the existing structure, releasing C₅₅PP as a by-product. The resultant C₅₅PP must be recycled to C₅₅P before being used in another round of cell wall polymer biosynthesis. The major protein responsible for recycling in Escherichia coli is undecaprenyl pyrophosphate phosphatase (UppP). The ongoing synthesis and recycling of undecaprenyl phosphate by UppS and UppP, respectively, are required for the survival and pathogenesis of bacteria; thus, both enzymes represent attractive targets for the development of therapeutics. In this thesis, UppP was structurally and functionally characterized, and inhibition of UppS by novel inhibitors was investigated. The X-ray crystallographic structure of the polytopic integral membrane protein membrane protein UppP was solved to 2.0 Å resolution using lipid cubic phase (LCP) crystallization. The crystal structure revealed an unexpected membrane topology and three-dimensional structure that suggests a potential role for UppP as a C₅₅P(P) lipid flippase and allowed for the rationalization of previously published site-directed mutagenesis results. An ordered monoolein molecule in the active site of the enzyme allowed us to model a C₅₅PP and propose a catalytic mechanism for C₅₅PP dephosphorylation. The crystal structure of UppS from Bacillus subtilis was solved in apo- and inhibitor bound states using X-ray crystallography, allowing us to rationalize two novel inhibitors’ superior efficacy against B. subtilis UppS versus Staphylococcus aureus or E. coli orthologues. The inhibitors bind in the hydrophobic tunnel into which the nascent C55PP product of UppS grows. Additionally, a crystal structure of B. subtilis UppS in complex with clomiphene provided a clearer structural basis of its inhibition of UppS and provides a basis for the rational design of improved UppS inhibitors.

Text

2021-10-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/74337

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/