Taxonomic group: bacteria / Proteobacteria (Phylum: Proteobacteria)
Associated disease: infection due to Escherichia coli [ICD11: XN6P4 ]
 
NCBI PubMed ID: 18422345
Publication DOI: 10.1021/bi800240r
Journal NLM ID: 0370623
Publisher: American Chemical Society
Correspondence: raetzbiochem.duke.edu
Institutions: Department of Biochemistry, Duke University Medical Center, P.O. Box 3711, Durham, North Carolina 27710, USA

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

X-ray, Escherichia coli, lipid A, Chlamydia trachomatis, kinetics, acyltransferases

Structure type: oligomer ; 2237.336
Location inside paper: p.5291, fig.1A, Kdo2-lipid A
Compound class: lipid A
Contained glycoepitopes: IEDB_130650,IEDB_130659,IEDB_135394,IEDB_135515,IEDB_141807,IEDB_150908,IEDB_151531,IEDB_176772,IEDB_534864
 
Methods: X-ray, SDS-PAGE, DNA techniques, kinetics assays, genetic methods, biochemical methods, enzyme assay
Biological activity: kinetic data of LpxD acyltransferase
3D data: 3D data
 
NCBI Taxonomy refs (TaxIDs): 562
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