Steady-State Kinetics and Mechanism of LpxD, the N-Acyltransferase of Lipid A Biosynthesis^†

LpxD catalyzes the third step of lipid A biosynthesis, the (R)-3-hydroxymyristoyl-acyl carrier protein (R-3-OHC₁₄-ACP)-dependent N-acylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-α-d-glucosamine [UDP-3-O-(R-3-OHC₁₄)-GlcN]. We have now overexpressed and purified Escherichia coli LpxD to homogeneity. Steady-state kinetics suggest a compulsory ordered mechanism in which R-3-OHC₁₄-ACP binds prior to UDP-3-O-(R-3-OHC₁₄)-GlcN. The product, UDP-2,3-diacylglucosamine, dissociates prior to ACP; the latter is a competitive inhibitor against R-3-OHC₁₄-ACP and a noncompetitive inhibitor against UDP-3-O-(R-3-OHC₁₄)-GlcN. UDP-2-N-[(R)-3-Hydroxymyristoyl]-α-d-glucosamine, obtained by mild base hydrolysis of UDP-2,3-diacylglucosamine, is a noncompetitive inhibitor against both substrates. Synthetic (R)-3-hydroxylauroyl-methylphosphopantetheine is an uncompetitive inhibitor against R-3-OHC₁₄-ACP and a competitive inhibitor against UDP-3-O-(R-3-OHC₁₄)-GlcN, but (R)-3-hydroxylauroyl-methylphosphopantetheine is also a very poor substrate. A compulsory ordered mechanism is consistent with the fact that R-3-OHC₁₄-ACP has a high binding affinity for free LpxD whereas UDP-3-O-(R-3-OHC₁₄)-GlcN does not. Divalent cations inhibit R-3-OHC₁₄-ACP-dependent acylation but not (R)-3-hydroxylauroyl-methylphosphopantetheine-dependent acylation, indicating that the acidic recognition helix of R-3-OHC₁₄-ACP contributes to binding. The F41A mutation increases the K_M for UDP-3-O-(R-3-OHC₁₄)-GlcN 30-fold, consistent with aromatic stacking of the corresponding F43 side chain against the uracil moiety of bound UDP-GlcNAc in the X-ray structure of Chlamydia trachomatis LpxD. Mutagenesis implicates E. coli H239 but excludes H276 as the catalytic base, and neither residue is likely to stabilize the oxyanion intermediate.