Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis.
The MraY translocase catalyzes the first membrane step of bacterial cell wall peptidoglycan synthesis (i.e. the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid), a reversible reaction yielding undecaprenylpyrophosphoryl-N-acetylmuramoyl-pentapeptide (lipid intermediate I). This essential integral membrane protein, which is considered as a very promising target for the search of new antibacterial compounds, has thus far been clearly underexploited due to its intrinsic refractory nature to overexpression and purification. We here report conditions for the high level overproduction and for the first time the purification to homogeneity of milligram quantities of MraY protein. The kinetic parameters and effects of pH, salts, cations, and detergents on enzyme activity are described, taking the Bacillus subtilis MraY translocase as a model.
Bouhss
Ahmed
A
Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, UMR 8619 CNRS, Université Paris-Sud, Bât. 430, 91405 Orsay, France. ahmed.bouhss@ebp.u-psud.fr
Crouvoisier
Muriel
M
Blanot
Didier
D
Mengin-Lecreulx
Dominique
D
D020816:Amino Acid Motifs; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D010457:Peptidoglycan / Q000096:biosynthesis* / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012492:Salts / Q000737:chemistry / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014166:Transferases / Q000737:chemistry* / Q000302:isolation & purification*; D017855:Transferases (Other Substituted Phosphate Groups); D014415:Tunicamycin / Q000494:pharmacology; D014530:Uridine Diphosphate / Q000737:chemistry
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002412:Cations; D003902:Detergents; D008055:Lipids; D010455:Peptides; D010457:Peptidoglycan; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012492:Salts; D014415:Tunicamycin; D014530:Uridine Diphosphate; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria