Enzymatic properties and substrate specificity of a bacterial phosphatidylcholine synthase. (14th July 2014)

• Record Type: Journal Article
• Authors: Aktas, Meriyem; Köster, Stefan; Kizilirmak, Sarah; Casanova, Javier C.; Betz, Heidi; Fritz, Christiane; Moser, Roman; Yildiz, Özkan; Narberhaus, Franz
• Is Part Of: FEBS journal. Volume 281:Number 15(2014)
• Page Start: 3523
• Page End: 3541
• Publication Date: 2014-07-14
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.12877
• Languages: English
• ISSNs: 1742-464X

Abstract

<abstract abstract-type="main" id="febs12877-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="febs12877-sec-0001" sec-type="section"> <p>Phosphatidylcholine (PC) is a rare membrane lipid in bacteria, but is crucial for virulence of the plant pathogen <italic>Agrobacterium tumefaciens</italic> and various other pathogens. <italic>Agrobacterium tumefaciens</italic> 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 (DGX<sub>2</sub>ARX<sub>12</sub>GX<sub>3</sub>DX<sub>3</sub>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.</p> </sec> <sec id="febs12877-sec-0002" sec-type="section"> <title>Structured digital abstract</title> <p> <list id="febs12877-list-0001" list-type="bullet"> <list-item> <p> <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/A9CIM3" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">Pcs</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/A9CIM3" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">Pcs</ext-link> <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">bind</ext-link> by <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">molecular sieving</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9549401" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">1</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9549426" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">2</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9549420" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">3</ext-link>)</p> </list-item> </list> </p> </sec> </abstract>