1. Enzymatic properties and substrate specificity of a bacterial phosphatidylcholine synthase.
- Author
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Aktas M, Köster S, Kizilirmak S, Casanova JC, Betz H, Fritz C, Moser R, Yildiz Ö, and Narberhaus F
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Bacterial Proteins genetics, Bacterial Proteins metabolism, Carnitine chemistry, Catalytic Domain, Choline chemistry, Escherichia coli, Molecular Sequence Data, Protein Binding, Protein Structure, Quaternary, Substrate Specificity, Transferases (Other Substituted Phosphate Groups) genetics, Transferases (Other Substituted Phosphate Groups) metabolism, Agrobacterium tumefaciens enzymology, Bacterial Proteins chemistry, Transferases (Other Substituted Phosphate Groups) chemistry
- Abstract
Phosphatidylcholine (PC) is a rare membrane lipid in bacteria, but is crucial for virulence of the plant pathogen Agrobacterium tumefaciens and various other pathogens. Agrobacterium tumefaciens uses two independent PC biosynthesis pathways. One is dependent on the integral membrane protein PC synthase (Pcs), which catalyzes the conversion of cytidine diphosphate-diacylglycerol (CDP-DAG) and choline to PC, thereby releasing a cytidine monophosphate (CMP). Here, we show that Pcs consists of eight transmembrane segments with its N- and C-termini located in the cytoplasm. A cytoplasmic loop between the second and third membrane helix contains the majority of the conserved amino acids of a CDP-alcohol phosphotransferase motif (DGX2 ARX12 GX3 DX3 D). Using point mutagenesis, we provide evidence for a crucial role of this motif in choline binding and enzyme activity. To study the catalytic features of the enzyme, we established a purification protocol for recombinant Pcs. The enzyme forms stable oligomers and exhibits broad substrate specificity towards choline derivatives. The presence of CDP-DAG and manganese is a prerequisite for cooperative binding of choline. PC formation by Pcs is reversible and proceeds via two successive reactions. In a first choline- and manganese-independent reaction, CDP-DAG is hydrolyzed releasing a CMP molecule. The resulting phosphatidyl intermediate reacts with choline in a second manganese-dependent step to form PC., Structured Digital Abstract: Pcs and Pcs bind by molecular sieving (1, 2, 3)., (© 2014 FEBS.)
- Published
- 2014
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