title: Active site mapping of MraY, a member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, catalyzing the first membrane step of peptidoglycan biosynthesis.
abstract: The MraY transferase is an integral membrane protein that catalyzes an essential step of peptidoglycan biosynthesis, namely the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid. It belongs to a large superfamily of eukaryotic and prokaryotic prenyl sugar transferases. No 3D structure has been reported for any member of this superfamily, and to date MraY is the only protein that has been successfully purified to homogeneity. Nineteen polar residues located in the five cytoplasmic segments of MraY appeared as invariants in the sequences of MraY orthologues. A certain number of these invariant residues were found to be conserved in the whole superfamily. To assess the importance of these residues in the catalytic process, site-directed mutagenesis was performed using the Bacillus subtilis MraY as a model. Fourteen residues were shown to be essential for MraY activity by an in vivo functional complementation assay using a constructed conditional mraY mutant strain. The corresponding mutant proteins were purified and biochemically characterized. None of these mutations did significantly affect the binding of the nucleotidic and lipidic substrates, but the k cat was dramatically reduced in almost all cases. The important residues for activity therefore appeared to be distributed in all the cytoplasmic segments, indicating that these five regions contribute to the structure of the catalytic site. Our data show that the D98 residue that is invariant in the whole superfamily should be involved in the deprotonation of the lipid substrate during the catalytic process.
authors.affiliation: Univ Paris-Sud, UMR 8619, Institut de Biochimie et de Biophysique Moleculaire et Cellulaire, Bat. 430, Orsay, France.
authors.forename: Bayan
authors.initials: B
authors.lastname: Al-Dabbagh
authors.forename: Xavier
authors.initials: X
authors.lastname: Henry
authors.forename: Meriem
authors.initials: M
authors.lastname: El Ghachi
authors.forename: Geneviève
authors.initials: G
authors.lastname: Auger
authors.forename: Didier
authors.initials: D
authors.lastname: Blanot
authors.forename: Claudine
authors.initials: C
authors.lastname: Parquet
authors.forename: Dominique
authors.initials: D
authors.lastname: Mengin-Lecreulx
authors.forename: Ahmed
authors.initials: A
authors.lastname: Bouhss
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010457:Peptidoglycan / Q000096:biosynthesis*; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins; D010457:Peptidoglycan; D015636:Magnesium Chloride; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria