Academic Commons

Theses Doctoral

Structural and Functional Investigation of Bacterial Membrane Biosynthesis

Belcher Dufrisne, Meagan Leigh

Integral membrane enzymes contribute a unique repertoire to the cell, as they are capable of synthesizing products from substrates of different chemical character at the membrane-water interface. Membrane-embedded enzymes are often responsible for the synthesis of important components of the cellular membrane and contribute to the structural integrity of the cell, maintenance of cellular homeostasis and signal transduction. One of the main focuses of Dr. Filippo Mancia’s laboratory is understanding how enzymes complete these functions by investigating, at an atomic level, the determinants of substrate binding and catalysis within the membrane and at the membrane surface. Here I will present my investigation of two such integral membrane enzyme systems, which are responsible for the synthesis and processing of membrane-embedded molecules in bacteria.
Phosphatidylinositol-phosphate Synthase (PIPS)
Phosphaitylinositol (PI) is an essential lipid component in mycobacteria, demonstrated by loss of viability when PI is reduced to 50% of wild-type levels. Phosphatidylinositol (PI) is required for the biosynthesis of key components of the cell wall, such as the glycolipids phosphatidylinositol-mannosides, lipomannan and lipoarabinomannan. For these molecules, PI serves as a common lipid anchor to the membrane. In Mycobacterium tuberculosis, the disease causing pathogen of tuberculosis, these glycolipids function as important virulence factors and modulators of the host immune response. Therefore, the enzyme responsible for PI synthesis in this organism is a potential target for the development of anti-tuberculosis drugs.
The defining step in phosphatidylinositol biosynthesis is catalyzed by a member of the CDP-alcohol phosphotransferase enzyme family. The enzyme uses CDP-diacylglycerol as the donor substrate, and either inositol in eukaryotes or inositol-phosphate in prokaryotes as the acceptor alcohol of the synthesis reaction. In prokaryotes, phosphatidylinositol-phosphate synthase (PIPS; a member of the CDP-alcohol phosphotransferase family) catalyzes this reaction to yield phosphatidylinositol-phosphate, which is then dephosphorylated to PI by an uncharacterized enzyme.
Structures of PIPS from Renibacterium salmoninarum (RsPIPS), with and without bound CDP-diacylglycerol, have revealed the location of the acceptor site as well as molecular determinants of substrate specificity and catalysis of the enzyme. However, RsPIPS has low activity relative to PIPS from Mycobacterium tuberculosis (MtPIPS) and the two share only 40% protein sequence identity. Therefore, these initial structures have limited potential for meaningful homology modeling and drug design. Presented here are the structures of PIPS from Mycobacterium kansasii (MkPIPS), which is 86% identical to MtPIPS, in an apo state to 3.1 Å resolution, in a nucleotide-bound state to 3.5 Å resolution, and in a novel ligand-bound state to 2.6 Å resolution. This work provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens.
RodA-PBP2 Complex
The cell wall of most gram-negative and gram-positive bacteria (excluding atypical bacteria such as members of Mycoplasmataceae) is composed of peptidoglycan, a mesh of repeating carbohydrates (N-acetylmuramic acid, MurNAc, and N-acetylglucosamine, GlcNAc) cross-linked by small peptides. Peptidoglycan is essential for growth, division and viability of the organism. Any disruption of the biosynthesis of peptidoglycan, whether by genetic mutation, inhibition with antibiotics or degradation by lysozyme, results in bacterial cell lysis. Peptidoglycan helps maintain cell shape and serves as an anchor for accessory proteins and other cell wall components. As essential components of the cell wall, enzymes contributing to the peptidoglycan biosynthetic pathway can be exploited as antibiotic targets.
After a hydrophilic peptidoglycan precursor (UDP-MurNAc-pentapeptide) is synthesized in the cytosol, it is attached to the lipid carrier undecaprenyl phosphate (UndP). The lipid-linked precursor (undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide or Lipid I) is modified further to undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc (Lipid II) by addition of a GlcNAc moiety. Lipid II is then flipped across the membrane to the periplasm where its sugars are polymerized to form the glycan strands of the peptidoglycan mesh. SEDS proteins, essential for maintaining bacterial processes that determine shape, elongation, cell division and sporulation, are integral membrane enzyme that have been implicated in this process as either Lipid II flippases, glycosyltransferases responsible for sugar polymerization, or both. SEDS proteins are also known to form a functional complex with type b penicillin-binding proteins (PBPs), which are known as transpeptidase enzymes, responsible for the crosslinking of peptides in the formation of the peptidoglycan mesh.
Though structures of both RodA (a SEDS protein involved in bacterial growth and elongation) and type b PBPs are available, the interaction between the two proteins and their joint enzymatic activity is poorly characterized. Here, I present the preliminary structural characterization of a RodA-PBP2 protein complex by single-particle cryo-electron microscopy (cryo-EM). We hope this ongoing work will contribute to the understanding of these enzymes and to the development of antibiotics to combat antibiotic resistance.

Files

This item is currently under embargo. It will be available starting 2019-10-05.

More About This Work

Academic Units
Physiology and Cellular Biophysics
Thesis Advisors
Mancia, Filippo
Degree
Ph.D., Columbia University
Published Here
October 9, 2018
Academic Commons provides global access to research and scholarship produced at Columbia University, Barnard College, Teachers College, Union Theological Seminary and Jewish Theological Seminary. Academic Commons is managed by the Columbia University Libraries.