FIGURE 7.
Charge deconvoluted ESI FT-ICR mass spectra in negative ion mode demonstrating the WaaAAAE-catalyzed conversion of the tetraacyl-1,4′-bisphosphate lipid A precursor 406 (1404.85 u) (A) into Kdo-406 (1624.91 u) without (B) and with treatment of the Kdo transferase at 80 °C for 16 h (C) prior to the determination of enzymatic activity. The activity was assayed in reaction mixtures containing 100 mm HEPES, pH 7.5, 10% glycerol, 10 mm MgCl2, 2 mm Kdo, 5 mm CTP, 3.2 mm Triton X-100, and 107 μm compound 406. The reactions were incubated at 60 °C for 1 h with no enzymes (A) and in the presence of 17 milliunits of purified KdsBAAE and 75 μm either untreated (B) or pretreated WaaAAAE (C). The peak at 1320.60 u is consistent with a batch-dependent byproduct of 406 synthesis (A). The peaks at 1178.66 u (A) and 1398.72 u (B and C) are probably triacylated degradation products of compound 406 and Kdo-406 produced during sample preparation, respectively.