Enzymatic properties and substrate specificity of a bacterial phosphatidylcholine synthase

被引:14
作者
Aktas, Meriyem [1 ]
Koester, Stefan [2 ]
Kizilirmak, Sarah [1 ]
Casanova, Javier C. [2 ]
Betz, Heidi [2 ]
Fritz, Christiane [1 ]
Moser, Roman [1 ]
Yildiz, Oezkan [2 ]
Narberhaus, Franz [1 ]
机构
[1] Ruhr Univ Bochum, Dept Microbial Biol, D-44780 Bochum, Germany
[2] Max Planck Inst Biophys, Dept Biol Struct, Frankfurt, Germany
关键词
Agrobacterium tumefaciens; CDP-alcohol phosphotransferase; phosphatidylcholine; phosphatidylcholine synthase; phospholipids; ESCHERICHIA-COLI; AGROBACTERIUM-TUMEFACIENS; SACCHAROMYCES-CEREVISIAE; SYNTHESIZES PHOSPHATIDYLCHOLINE; PHOSPHATIDYLSERINE SYNTHETASE; NEGATIVE COOPERATIVITY; PHOSPHOLIPID-SYNTHESIS; MEMBRANE-PROTEINS; PURIFICATION; CHOLINE;
D O I
10.1111/febs.12877
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (DGX(2)ARX(12)GX(3)DX(3)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.
引用
收藏
页码:3523 / 3541
页数:19
相关论文
共 59 条
[1]   Membrane lipids in Agrobacterium tumefaciens: biosynthetic pathways and importance for pathogenesis [J].
Aktas, Meriyem ;
Danne, Linna ;
Moeller, Philip ;
Narberhaus, Franz .
FRONTIERS IN PLANT SCIENCE, 2014, 5
[2]   Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter [J].
Aktas, Meriyem ;
Jost, Kathinka A. ;
Fritz, Christiane ;
Narberhaus, Franz .
JOURNAL OF BACTERIOLOGY, 2011, 193 (19) :5119-5129
[3]   S-Adenosylmethionine-Binding Properties of a Bacterial Phospholipid N-Methyltransferase [J].
Aktas, Meriyem ;
Gleichenhagen, Jan ;
Stoll, Raphael ;
Narberhaus, Franz .
JOURNAL OF BACTERIOLOGY, 2011, 193 (14) :3473-3481
[4]   Phosphatidylcholine biosynthesis and its significance in bacteria interacting with eukaryotic cells [J].
Aktas, Meriyem ;
Wessel, Mirja ;
Hacker, Stephanie ;
Kluesener, Sonja ;
Gleichenhagen, Jan ;
Narberhaus, Franz .
EUROPEAN JOURNAL OF CELL BIOLOGY, 2010, 89 (12) :888-894
[5]  
BAELEE MS, 1984, J BIOL CHEM, V259, P857
[6]  
BLIGH EG, 1959, CAN J BIOCHEM PHYS, V37, P911
[7]   The selective utilization of substrates in vivo by the phosphatidylethanolamine and phosphatidylcholine biosynthetic enzymes Ept1p and Cpt1p in yeast [J].
Boumann, HA ;
de Kruijff, B ;
Heck, AJR ;
de Kroon, AIPM .
FEBS LETTERS, 2004, 569 (1-3) :173-177
[8]   FORMATION OF PHOSPHOLIPIDS CONTAINING UNNATURAL BASES BY CYTIDINE PATHWAY [J].
CHOJNACKI, T ;
ANSELL, GB .
JOURNAL OF NEUROCHEMISTRY, 1967, 14 (04) :413-+
[9]  
CLAROS MG, 1994, COMPUT APPL BIOSCI, V10, P685