Purification and characterization of the bacterial UDP-GlcNAc:undecaprenyl-phosphate GlcNAc-1-phosphate transferase WecA.
To date, the structural and functional characterization of proteins belonging to the polyprenyl-phosphate N-acetylhexosamine-1-phosphate transferase superfamily has been relentlessly held back by problems encountered with their overexpression and purification. In the present work and for the first time, the integral membrane protein WecA that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate, yielding undecaprenyl-pyrophosphoryl-GlcNAc, the lipid intermediate involved in the synthesis of various bacterial cell envelope components, was overproduced and purified to near homogeneity in milligram quantities. An enzymatic assay was developed, and the kinetic parameters of WecA as well as the effects of pH, salts, cations, detergents, and temperature on the enzyme activity were determined. A minimal length of 35 carbons was required for the lipid substrate, and tunicamycin was shown to inhibit the enzyme at submicromolar concentrations.
Al-Dabbagh
Bayan
B
Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619 CNRS, Université Paris-Sud, Bât. 430, 91405 Orsay Cedex, France.
Mengin-Lecreulx
Dominique
D
Bouhss
Ahmed
A
D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D012492:Salts / Q000494:pharmacology; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D029968:Escherichia coli Proteins; D012492:Salts; D014415:Tunicamycin; D017855:Transferases (Other Substituted Phosphate Groups); C083617:wecA protein, E coli