Kinetic analysis of the zinc-dependent deacetylase in the lipid A biosynthetic pathway.
The first committed step of lipid A biosynthesis in Gram-negative bacteria is catalyzed by the zinc-dependent hydrolase 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 orthologue that displays 32% sequence identity to LpxC from Escherichia coli. The detailed reaction mechanism and specific roles of active site residues for LpxC from A. aeolicus are further analyzed here. The pH dependencies of k(cat)/K(M) and k(cat) for the deacetylation of the substrate UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc are both bell-shaped. The ascending acidic limb (pK(1)) was fitted to 6.1 +/- 0.2 for k(cat) and 5.7 +/- 0.2 for k(cat)/K(M). The descending basic limb (pK(2)) was fitted to 8.0 +/- 0.2 for k(cat) and 8.4 +/- 0.2 for k(cat)/K(M). The pH dependence of the E73A mutant exhibits loss of the acidic limb, and the mutant retains only 0.15% activity versus the wild type. The pH dependencies of the other active site mutants H253A, K227A, H253A/K227A, and D234N remain bell-shaped, although their significantly lower activities (0.25%, 0.05%, 0.007%, and 0.57%, respectively) suggest 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.
McClerren
Amanda L
AL
Department of Biochemistry, Duke University Medical Center, P.O. Box 3813, Durham, North Carolina 27710, USA.
Zhou
Pei
P
Guan
Ziqiang
Z
Raetz
Christian R H
CR
Rudolph
Johannes
J
D000409:Alanine / Q000235:genetics; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008050:Lipid A / Q000096:biosynthesis*; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity / Q000235:genetics; D015032:Zinc / Q000737:chemistry*
D001426:Bacterial Proteins; D008050:Lipid A; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D001216:Asparagine; D000581:Amidohydrolases; C097307:UDP-3-O-acyl-N-acetylglucosamine deacetylase; D015032:Zinc; D008239:Lysine; D000409:Alanine