Enzymatic properties and substrate specificity of a bacterial phosphatidylcholine synthase


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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 (DGX2ARX12GX3DX3D). 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 abstractPcs and Pcs bind by molecular sieving (1, 2, 3)The integral membrane protein phosphatidylcholine (PC) synthase (Pcs) converts cytidine diphosphate-diacylglycerol (CDP-DAG) and choline to PC. Here we describe the first purification of active recombinant Pcs, which exhibits broad specificity towards choline derivatives. In vitro enzyme assays suggest that Pcs catalyzes a reversible two-step reaction with CDP-DAG hydrolysis and cytidine monophosphate release followed by transfer of choline to the phosphatidyl-enzyme intermediate.

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