Extended Data Figure 5: Specific activity of wild-type and mutant MraYAA in the presence and absence of MD2.
From: Structural insights into inhibition of lipid I production in bacterial cell wall synthesis
a, Normalized specific activity of wild-type (WT) MraYAA and enzymatically active mutants with and without MD2 treatment. Wild-type MraYAA or mutant MraYAA was added to the reaction mixture to a final concentration that enabled product detection within the enzymatic linear range: 50 nM (WT), 500 nM (Lys70Ala), 400 nM (Thr75Ala), 350 nM (Asp193Asn), 250 nM (Asn255Ala), 200 nM (Phe262Ala), 50 nM (Phe262Trp), 400 nM (Gln305Ala), and 500 nM (His325Ala). Each reaction was carried out in the presence of either 0 μM, 0.3 μM or 1 μM MD2. Data are shown for three technical replicates ± s.e.m. Specific activity measurements for each mutant were normalized relative to that without added MD2. b, Specific activity of wild-type MraYAA and enzymatically inactive mutants. MraYAA Asn190Ala, Asp193Ala, Asp196Ala and Asp196Asn were each added to a final concentration of 500 nM, while wild-type MraYAA was present at 50 nM. All enzymatic reactions were conducted with a radiochemical assay monitoring the transfer of [14C]phospho-MurNAc-pentapeptide from [14C]UM5A to C55-P, forming [14C]lipid I. The radiolabelled product, [14C]lipid I, was quantified using a liquid scintillation counting method (d.p.m.). Specific activity was calculated by determining moles of [14C]lipid I formed, divided by the reaction time and the quantity of enzyme added. Three technical replicates are shown with the mean value indicated by a line.
