Saltar al contenido
Merck

Enzymatic properties and substrate specificity of a bacterial phosphatidylcholine synthase.

The FEBS journal (2014-06-17)
Meriyem Aktas, Stefan Köster, Sarah Kizilirmak, Javier C Casanova, Heidi Betz, Christiane Fritz, Roman Moser, Özkan Yildiz, Franz Narberhaus
RESUMEN

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. Pcs and Pcs bind by molecular sieving (1, 2, 3).

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
Acetonitrilo, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Ácido acético, glacial, ACS reagent, ≥99.7%
Sigma-Aldrich
Fosfato de potasio monobasic, ACS reagent, ≥99.0%
Sigma-Aldrich
Acetonitrilo, HPLC Plus, ≥99.9%
Sigma-Aldrich
Ácido acético, glacial, ReagentPlus®, ≥99%
Sigma-Aldrich
Cloroformo, contains 100-200 ppm amylenes as stabilizer, ≥99.5%
Sigma-Aldrich
Cloroformo, HPLC Plus, for HPLC, GC, and residue analysis, ≥99.9%, contains amylenes as stabilizer
Sigma-Aldrich
Cloroformo, suitable for HPLC, ≥99.8%, contains 0.5-1.0% ethanol as stabilizer
Sigma-Aldrich
1-Propanol, suitable for HPLC, ≥99.9%
Sigma-Aldrich
Acetonitrilo, ACS reagent, ≥99.5%
Sigma-Aldrich
Cloroformo, contains ethanol as stabilizer, ACS reagent, ≥99.8%
Sigma-Aldrich
Acetonitrilo, anhydrous, 99.8%
Sigma-Aldrich
Cloruro de colina, ≥98%
Sigma-Aldrich
Fosfato de potasio monobasic, powder, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0%
Sigma-Aldrich
Acetonitrilo, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Ácido acético, glacial, ≥99.99% trace metals basis
Sigma-Aldrich
1-Propanol, ACS reagent, ≥99.5%
Sigma-Aldrich
Ácido acético solution, suitable for HPLC
Sigma-Aldrich
Ácido acético, glacial, puriss., meets analytical specification of Ph. Eur., BP, USP, FCC, 99.8-100.5%
Sigma-Aldrich
Cloroformo, suitable for HPLC, ≥99.8%, amylene stabilized
Sigma-Aldrich
Fosfato de potasio monobasic, buffer substance, anhydrous, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., 99.5-100.5%
Sigma-Aldrich
Ácido acético, glacial, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.8%
Sigma-Aldrich
Cloroformo, contains amylenes as stabilizer, ACS reagent, ≥99.8%
Sigma-Aldrich
Cloroformo, ReagentPlus®, ≥99.8%, contains 0.5-1.0% ethanol as stabilizer
Sigma-Aldrich
Cloroformo, puriss. p.a., reag. ISO, reag. Ph. Eur., 99.0-99.4% (GC)
Sigma-Aldrich
Cloruro de colina, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥98%
Sigma-Aldrich
2-Dimethylaminoethanol, ≥99.5%
Sigma-Aldrich
Fosfato de potasio monobasic, for molecular biology, ≥98.0%
Sigma-Aldrich
Acetonitrilo, suitable for HPLC-GC, ≥99.8% (GC)
Sigma-Aldrich
Cloroformo, HPLC Plus, for HPLC, GC, and residue analysis, ≥99.9%, contains 0.5-1.0% ethanol as stabilizer