Extended Figure 1.

MraY catalyzes the formation of Lipid I and binds MD2. a, Scheme of the reaction catalyzed by MraY. The U-labeled blue hexagon represents uridine and the M-labeled orange hexagon represents MurNAc. The phosphates associated with the lipid carrier C₅₅-P are shown as red circles, and the phosphates from the substrate, UDP-MurNAc-pentapeptide (UM5A), are shown as yellow circles. b, Chemical structures of the substrate, UM5A (top) and the inhibitor MD2 (bottom). c, Michaelis-Menten kinetic characterization of MraY_AA translocase activity. The reaction monitored is the MraY_AA-catalyzed transfer of [¹⁴C]phospho-MurNAc-pentapeptide from [¹⁴C]UM5A to C₅₅-P, forming [¹⁴C]Lipid I. The enzymatic parameters measured are as follows: K_M = 190 ± 60 μM, k_cat = 20 ± 2 min⁻¹, k_cat/K_M = 0.11 ± 0.3 μM⁻¹ min⁻¹. Data are shown as means of three technical replicates ± s.e.m. d, MD2 (green) in complex with MraY_AA. The distances between MD2 and the three catalytic acidic residues D117, D118 and D265 (magenta) are all greater than 4.5 Å.