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]-alpha-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]-alpha-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.
Bartling
Craig M
CM
Department of Biochemistry, Duke University Medical Center, P.O. Box 3711, Durham, North Carolina 27710, USA.
Raetz
Christian R H
CR
D000213:Acyl Carrier Protein / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D002692:Chlamydia trachomatis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008050:Lipid A / Q000096:biosynthesis*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D040681:Structural Homology, Protein
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008024:Ligands; D008050:Lipid A; D011994:Recombinant Proteins; D000217:Acyltransferases