Structural basis for catalysis and selectivity of phospholipid synthesis by eukaryotic choline-phosphotransferase

Jacquelyn R Roberts; Yasuhiro Horibata; Frank E Kwarcinski; Vinson Lam; Ashleigh M Raczkowski; Akane Hubbard; Betsy White; Hiroyuki Sugimoto; Gregory G Tall; Melanie D Ohi; Shoji Maeda

doi:10.1038/s41467-024-55673-1

Structural basis for catalysis and selectivity of phospholipid synthesis by eukaryotic choline-phosphotransferase

Nat Commun. 2025 Jan 2;16(1):111. doi: 10.1038/s41467-024-55673-1.

Authors

Jacquelyn R Roberts^#^{1

2}, Yasuhiro Horibata^#³, Frank E Kwarcinski^#⁴, Vinson Lam¹, Ashleigh M Raczkowski¹, Akane Hubbard⁵, Betsy White⁶, Hiroyuki Sugimoto³, Gregory G Tall⁴, Melanie D Ohi^{7

8}, Shoji Maeda^{9

10}

Affiliations

¹ Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.
² Department of Biological Chemistry, University of Michigan School of Medicine, Ann Arbor, MI, USA.
³ Department of Biochemistry, Dokkyo Medical University School of Medicine, Mibu, Tochigi, Japan.
⁴ Department of Pharmacology, University of Michigan School of Medicine, Ann Arbor, MI, USA.
⁵ Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, USA.
⁶ Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.
⁷ Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA. [email protected].
⁸ Department of Cell and Developmental Biology, University of Michigan School of Medicine, Ann Arbor, MI, USA. [email protected].
⁹ Department of Pharmacology, University of Michigan School of Medicine, Ann Arbor, MI, USA. [email protected].
¹⁰ Xaira Therapeutics, Brisbane, CA, USA. [email protected].

^# Contributed equally.

Abstract

Phospholipids are the most abundant component in lipid membranes and are essential for the structural and functional integrity of the cell. In eukaryotic cells, phospholipids are primarily synthesized de novo through the Kennedy pathway that involves multiple enzymatic processes. The terminal reaction is mediated by a group of cytidine-5'-diphosphate (CDP)-choline /CDP-ethanolamine-phosphotransferases (CPT/EPT) that use 1,2-diacylglycerol (DAG) and CDP-choline or CDP-ethanolamine to produce phosphatidylcholine (PC) or phosphatidylethanolamine (PE) that are the main phospholipids in eukaryotic cells. Here we present the structure of the yeast CPT1 in multiple substrate-bound states. Structural and functional analysis of these binding-sites reveal the critical residues for the DAG acyl-chain preference and the choline/ethanolamine selectivity. Additionally, we present the structure in complex with a potent inhibitor characterized in this study. The ensemble of structures allows us to propose the reaction mechanism for phospholipid biosynthesis by the family of CDP-alcohol phosphotransferases (CDP-APs).

MeSH terms

Binding Sites
Biocatalysis
Catalysis
Crystallography, X-Ray
Cytidine Diphosphate / analogs & derivatives
Cytidine Diphosphate Choline / chemistry
Cytidine Diphosphate Choline / metabolism
Diglycerides / biosynthesis
Diglycerides / chemistry
Diglycerides / metabolism
Ethanolamines
Models, Molecular
Phosphatidylcholines / biosynthesis
Phosphatidylcholines / chemistry
Phosphatidylcholines / metabolism
Phosphatidylethanolamines / biosynthesis
Phosphatidylethanolamines / chemistry
Phosphatidylethanolamines / metabolism
Phospholipids* / biosynthesis
Phospholipids* / chemistry
Phospholipids* / metabolism
Phosphotransferases (Alcohol Group Acceptor) / chemistry
Phosphotransferases (Alcohol Group Acceptor) / metabolism
Saccharomyces cerevisiae Proteins / chemistry
Saccharomyces cerevisiae Proteins / genetics
Saccharomyces cerevisiae Proteins / metabolism
Saccharomyces cerevisiae* / metabolism
Substrate Specificity

Substances

Phospholipids
Phosphatidylethanolamines
Cytidine Diphosphate Choline
Saccharomyces cerevisiae Proteins
Diglycerides
Phosphatidylcholines
CDP ethanolamine
Phosphotransferases (Alcohol Group Acceptor)
Cytidine Diphosphate
Ethanolamines

Abstract

MeSH terms

Substances

Grants and funding