MALDI-TOF MS identification of LPS-lipid A structure suggests different roles of putative cavity-forming sites in catalysis mechanism of ICR-Mo (AXE82_07515).

<p>MALDI-TOF MS profile of the LPS-lipid A pool isolated from the two negative controls, <i>E</i>. <i>coli</i> strain MG1655 alone (<b>A</b>) and with the empty vector pBAD24 (<b>B</b>). <b>C.</b> Appearance of a unique peak of the mono-modified lipid A, PPEA-1(or 4’)-lipid A, in the positive control, MG1655 strain carrying the wild-type of ICR-Mo (AXE82_07515). The two mutations of N110A (<b>D</b>) and T114A (<b>E</b>) failed to fully inactivate the enzymatic activity of ICR-Mo in that the modified peak, PPEA-lipid A is present. MALDI-TOF-MS analyses suggests that the three mutations of E118A (<b>F</b>), E246A (<b>G</b>) and T287A (<b>H</b>) impairs the function of ICR-Mo. The point-mutant (S332A) of ICR-Mo still possesses partial activity of catalyzing the transfer of PPEA to the 1(4’)-phosphate group of lipid A moieties. The six point-mutants of ICR-Mo are consistently inactive in the enzymatic activity of PEA transferase <i>in vivo</i>, including K335A (<b>J</b>), H397A (<b>K</b>), H402A (<b>L</b>), D472A (<b>M</b>), H473 (<b>N</b>) and H485A (<b>O</b>), respectively. The MS peak of lipid A species in <i>E</i>. <i>coli</i> is shown at m/z of 1796.744~1797.843, whereas its modified form appears at m/z of 1919.865~1920.457, when functional (even partial active) versions of ICR-Mo (AXE82_07515) are present in <i>E</i>. <i>coli</i>.