title: Functional and topological analysis of phosphatidylcholine synthase from Sinorhizobium meliloti.
abstract: Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (<20% of wild type activity) of Pcs activity. Six of these essential residues were subjected to further mutagenesis studies replacing them by amino acids with similar properties or size. A topological analysis of sinorhizobial Pcs showed the presence of eight transmembrane helices, with the C- and N-terminus located in the cytoplasm. The majority of the conserved residues is predicted to be either located within the cytoplasmic loops or on the cytoplasmic side of the membrane which can be expected for an enzyme using one membrane-associated and one soluble substrate.
authors.affiliation: Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Av. Universidad s/n, Apdo. Postal 565-A, Cuernavaca, Morelos, CP62210, Mexico.
authors.forename: Rosa L
authors.initials: RL
authors.lastname: Solís-Oviedo
authors.forename: Fernando
authors.initials: F
authors.lastname: Martínez-Morales
authors.forename: Otto
authors.initials: O
authors.lastname: Geiger
authors.forename: Christian
authors.initials: C
authors.lastname: Sohlenkamp
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D015153:Blotting, Western; D002794:Choline / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016962:Sinorhizobium meliloti / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C483298:CDP-diacylglycerol choline O-phosphatidyltransferase; D002794:Choline