X-ray structure of a CDP-alcohol phosphatidyltransferase membrane enzyme and insights into its catalytic mechanism

Przemyslaw Nogly; Ivan Gushchin; Alina Remeeva; Ana M. Esteves; Nuno Borges; Pik Yee Ma; Andrii Ishchenko; Sergei Grudinin; Ekaterina Round; Isabel Moraes; Valentin Borshchevskiy; Maria Helena Santos; Valentin Gordeliy; Margarida Archer Frazao

doi:10.1038/ncomms5169

X-ray structure of a CDP-alcohol phosphatidyltransferase membrane enzyme and insights into its catalytic mechanism

Przemyslaw Nogly, Ivan Gushchin, Alina Remeeva, Ana M. Esteves, Nuno Borges, Pik Yee Ma, Andrii Ishchenko, Sergei Grudinin, Ekaterina Round, Isabel Moraes, Valentin Borshchevskiy, Maria Helena Santos, Valentin Gordeliy, Margarida Archer Frazao

Instituto de Tecnologia Química e Biológica António Xavier (ITQB)

Research output: Contribution to journal › Article › peer-review

41 Citations (Scopus)

Abstract

Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65â ‰Å resolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane Î±-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed.

Original language	English
Article number	4169
Journal	Nature Communications
Volume	5
DOIs	https://doi.org/10.1038/ncomms5169
Publication status	Published - 19 Jun 2014

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Nogly, P., Gushchin, I., Remeeva, A., Esteves, A. M., Borges, N., Ma, P. Y., Ishchenko, A., Grudinin, S., Round, E., Moraes, I., Borshchevskiy, V., Santos, M. H., Gordeliy, V., & Frazao, M. A. (2014). X-ray structure of a CDP-alcohol phosphatidyltransferase membrane enzyme and insights into its catalytic mechanism. Nature Communications, 5, Article 4169. https://doi.org/10.1038/ncomms5169

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abstract = "Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65{\^a} ‰{\AA} resolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane {\^I}±-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed.",

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AU - Nogly, Przemyslaw

AU - Gushchin, Ivan

AU - Remeeva, Alina

AU - Esteves, Ana M.

AU - Borges, Nuno

AU - Ma, Pik Yee

AU - Ishchenko, Andrii

AU - Grudinin, Sergei

AU - Round, Ekaterina

AU - Moraes, Isabel

AU - Borshchevskiy, Valentin

AU - Santos, Maria Helena

AU - Gordeliy, Valentin

AU - Frazao, Margarida Archer

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AB - Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65â ‰Å resolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane Î±-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed.

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X-ray structure of a CDP-alcohol phosphatidyltransferase membrane enzyme and insights into its catalytic mechanism

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