Cellular membranes are critical components of all free-living organisms. However, knowledge of their biosynthesis and modification has been hindered by the hydrophobicity engendered by their lipid constituents. Lipids are synthesized and modified primarily by integral membrane enzymes embedded, at least in part, in the bilayer. However, the atomic-level details of lipid/enzyme interactions and the determinants of their specificity remain poorly understood. Here we present preliminary structural and functional characterization for three distinct families of integral membrane lipid-modifying enzymes. Two of the representative crystal structures have a bound lipid ligand, tripling the number of examples of membrane-embedded enzymes visualized with their cognate hydrophobic substrate. Each of the structures is a first representative of a large enzyme family: (1) GtrB, a polyisoprenyl phosphate glycosyltransferase attaches glucose to a lipid carrier for membrane translocation and a glycosyl donor for subsequent reactions. This reaction represents the first step in all protein glycosylation and glycosylation of the cell wall. (2) ArnT uses sugar-charged donors produced by GtrB-like enzymes, and transfers the saccharide to lipid A on the cell surface of bacteria, altering antibiotic resistance properties. (3) We present the structure of the Renibacterium salmoninarum phosphatidylinositol-phosphate (PIP) synthase - an enzyme required for inositol-lipid synthesis - with a bound CDP-diacylglycerol substrate. This enzyme is a member of the CDP-alcohol phosphotransferase family (CDP-APs), which catalyze the defining step in glycerophospholipid biosynthesis across all kingdoms of life. We will explore substrate recognition by these enzymes with a combination of experimental approaches including x-ray crystallography and structure-guided mutagenesis coupled to functional readouts in bacteria, yeast, and zebrafish. The overall goal of this proposal is to understand the basic principles of substrate recognition in lipid biosynthesis and modification reactions.

Public Health Relevance

Lipids are synthesized and modified primarily by enzymes embedded, at least in part, in the membrane bilayer, however, the atomic-level details of lipid/enzyme interactions and the determinants of their specificity remain poorly understood. We will use the knowledge gained from structures of three distinct families of integral membrane lipid-modifying enzymes to understand the basic principles of substrate recognition in lipid biosynthesis and modification. These data should reveal the molecular mechanisms of select human diseases, and provide pathways toward new classes of therapeutics targeting lipid/enzyme active sites.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM111980-02
Application #
9131765
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Chin, Jean
Project Start
2015-09-01
Project End
2019-08-31
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Physiology
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Dufrisne, Meagan Belcher; Petrou, Vasileios I; Clarke, Oliver B et al. (2017) Structural basis for catalysis at the membrane-water interface. Biochim Biophys Acta 1862:1368-1385
Scaglione, Antonella; Montemiglio, Linda Celeste; Parisi, Giacomo et al. (2017) Subcellular localization of the five members of the human steroid 5?-reductase family. Biochim Open 4:99-106
Petrou, Vasileios I; Herrera, Carmen M; Schultz, Kathryn M et al. (2016) Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation. Science 351:608-12
Chen, Yunting; Clarke, Oliver B; Kim, Jonathan et al. (2016) Structure of the STRA6 receptor for retinol uptake. Science 353:
Ardiccioni, Chiara; Clarke, Oliver B; Tomasek, David et al. (2016) Structure of the polyisoprenyl-phosphate glycosyltransferase GtrB and insights into the mechanism of catalysis. Nat Commun 7:10175
Clarke, Oliver B; Tomasek, David; Jorge, Carla D et al. (2015) Structural basis for phosphatidylinositol-phosphate biosynthesis. Nat Commun 6:8505