Kinetic analysis of the zinc-dependent deacetylase involved in lipid A biosynthesis
The first committed step of lipid A biosynthesis in Gram-negative bacteria is catalyzed by the Zn2+metalloenzyme LpxC that removes an acetate from the nitrogen at the 2 position of UDP-3-O-acyl- N-acetylglucosamine. Recent structural characterization by both NMR and X-ray crystallography provides many important details about the active site environment of LpxC from Aquifex aeolicus, a heat stable ortholog that displays 32% sequence identity to LpxC from Escherichia coli. The detailed reaction mechanism of LpxC, specific roles of its active site residues, and its inhibition by novel, small molecule compounds are investigated here.
The pH-dependence of kcat/KM and Kcat for the deacetylation of the substrate UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc by LpxC from A. aeolicus are both bell-shaped. The ascending acidic limb (pK1) was fitted to 6.1 ± 0.2 for kcat and 5.7 ± 0.2 for kcat/KM. The descending basic limb (pK2) was fitted to 8.0 ± 0.2 for kcat and 8.4 ± 0.2 for kcat/KM The pH-dependence of the E73A mutant exhibits loss of the acidic limb and retains only 0.15% activity versus wild type. The pH-dependence of the other active site mutants H253A, K227A, H253A/K227A, and D234N remains bell-shaped for each, although their significantly lower activity (0.25, 0.05, 0.007, and 0.57% each, respectively) suggests that they contribute significantly to catalysis. Our cumulative data support a mechanism for LpxC wherein Glu73 serves as the general-base for deprotonation and activation of the zinc-bound water.
In order to further evaluate the role of His253 in catalysis we have measured its pKa by NMR titration. Our results indicate that His253 is protonated in wild-type LpxC from A. aeolicus over the pH range of maximal catalysis, contributing a positive charge to the active site that could stabilize the oxyanion intermediate. We have also used this method to confirm that His253 is not responsible for either of the pH-dependent ionizations measured in the wild-type enzyme, in agreement with our kinetic analysis of the H253A mutant.
In addition we have examined the inhibition of LpxC activity by a new class of hydroxamate-containing compounds. These inhibitors have antibacterial properties against E. coli and Pseudomonas aeruginosa , comparable to ciprofloxacin. The mechanism of the most potent compound, IV-110, was investigated with LpxC from A. aeolicus and is time-dependent. IV-110 inhibits LpxC activity in two steps, the first of which is rapid and reversible. (Abstract shortened by UMI.)