Topological analysis of the MraY protein catalysing the first membrane step of peptidoglycan synthesis

The two-dimensional membrane topology of the Escherichia coli and Staphyloc occus aureus MraY transferases, which catalyse the formation of the first l ipid intermediate of peptidoglycan synthesis, was established using the bet a-lactamase fusion system. All 28 constructed mraY-blaM fusions produced hy brid proteins. Analysis of the ampicillin resistance of the strains with hy brids led to a common topological model possessing 10 transmembrane segment s, five cytoplasmic domains and six periplasmic domains including the N- an d C-terminal ends. The agreement between the topologies of E. coli and S. a ureus, their agreement to a fair extent with predicted models and a number of features arising from the comparative analysis of 25 orthologue sequence s strongly suggested the validity of the model for all eubacterial MraYs. T he primary structure of the 10 transmembrane segments diverged among orthol ogues, but they retained their hydrophobicity, number and size. The similar ity of the sequences and distribution of the five cytoplasmic domains in bo th models, as well as their conservation among the MraY orthologues, clearl y suggested their possible involvement in substrate recognition and in the catalytic process. Complementation tests showed that only fusions with untr uncated mraY restored growth. It was noteworthy that S. aureus MraY was fun ctional in E, coli. An increased MraY transferase activity was observed onl y with the untruncated hybrids from both organisms.