Topological analysis of the MraY protein catalysing the first membrane step of peptidoglycan synthesis.
The two-dimensional membrane topology of the Escherichia coli and Staphylococcus aureus MraY transferases, which catalyse the formation of the first lipid intermediate of peptidoglycan synthesis, was established using the beta-lactamase fusion system. All 28 constructed mraY-blaM fusions produced hybrid proteins. Analysis of the ampicillin resistance of the strains with hybrids led to a common topological model possessing 10 transmembrane segments, five cytoplasmic domains and six periplasmic domains including the N- and C-terminal ends. The agreement between the topologies of E. coli and S. aureus, their agreement to a fair extent with predicted models and a number of features arising from the comparative analysis of 25 orthologue sequences strongly suggested the validity of the model for all eubacterial MraYs. The primary structure of the 10 transmembrane segments diverged among orthologues, but they retained their hydrophobicity, number and size. The similarity of the sequences and distribution of the five cytoplasmic domains in both models, as well as their conservation among the MraY orthologues, clearly suggested their possible involvement in substrate recognition and in the catalytic process. Complementation tests showed that only fusions with untruncated mraY restored growth. It was noteworthy that S. aureus MraY was functional in E. coli. An increased MraY transferase activity was observed only with the untruncated hybrids from both organisms.
Bouhss
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A
Biochimie Structurale et Cellulaire, CNRS, Bâtiment 430, Université Paris-Sud, F-91405, Orsay, France.
Mengin-Lecreulx
D
D
Le Beller
D
D
Van Heijenoort
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D005816:Genetic Complementation Test; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010457:Peptidoglycan / Q000096:biosynthesis*; D019897:Periplasm / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology / Q000378:metabolism*; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D010457:Peptidoglycan; D011993:Recombinant Fusion Proteins; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria; D001618:beta-Lactamases