LpxD catalyzes the third step of lipid A biosynthesis, the R-3-hydroxymyristoyl-acyl carrier protein (R-3-OHC 14 -ACP)-dependent N-acylation of UDP-3-O-(R-3-hydroxymyristoyl)-α-D-glucosamine [UDP-3-O-(R-3-OHC 14 )-GlcN]. We have now over-expressed and purified E. coli LpxD to homogeneity. Steady state kinetics suggest a compulsory ordered mechanism in which R-3-OHC 14 -ACP binds prior to UDP-3-O-(R-3-OHC 14 )-GlcN. The product, UDP-2,3-diacylglucosamine, dissociates prior to ACP; the latter is a competitive inhibitor against R-3-OHC 14 -ACP and a noncompetitive inhibitor against UDP-3-O-(R-3-OHC 14 )-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-hydroxylauroylmethylphosphopantetheine is an uncompetitive inhibitor against R-3-OHC 14 -ACP and a competitive inhibitor against UDP-3-O-(R-3-OHC 14 )-GlcN, but R-3-hydroxylauroyl-methylphosphopantetheine is also a very poor substrate. A compulsory ordered mechanism is consistent with the fact that R-3-OHC 14 -ACP has a high binding affinity for free LpxD, whereas UDP-3-O-(R-3-OHC 14 )-GlcN does not. Divalent cations inhibit R-3-OHC 14 -ACP-dependent acylation but not R-3-hydroxylauroylmethylphosphopantetheine-dependent acylation, indicating that the acidic recognition helix of R-3-OHC 14 -ACP contributes to binding. The F41A mutation increases the K M for UDP-3-O-(R-3-OHC 14 )-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.Lipid A is the hydrophobic moiety of lipopolysaccharide (LPS) 1 , which constitutes the outer leaflet of the outer membrane of most Gram-negative bacteria (1-3). The lipid A moiety of LPS is usually required for bacterial growth (3,4) and is a potent activator of the mammalian innate immune system via the TLR4/MD-2 complex (5,6). Over-production of cytokines due to excessive stimulation of TLR4/MD-2 may occur during severe Gram-negative infections and may contribute to the life-threatening complications of septic shock (7,8).The Kdo 2 -lipid A substructure of Escherichia coli LPS is synthesized by a conserved system of nine constitutive enzymes (Fig. 1A) (3). LpxD catalyzes the third reaction in this scheme, the R-3-hydroxymyristoyl-acyl carrier protein (R-3-OHC 14 (Fig. 1A). Although essential for growth and an excellent target for the design of new antibiotics (9), LpxD is one of the least characterized enzymes in the pathway. Kelly and Raetz identified *Author to whom correspondence should be addressed: C. R. H. Raetz at (919) 684-3384; Fax (919) 684-8885; raetz@biochem.duke.edu. 1 The abbreviations are: ACP, acyl carrier protein; Bis-Tris, 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol; CMC, critical micelle concentration;...

LpxD catalyzes the third step of lipid A biosynthesis, the R-3-hydroxyacyl-ACP-dependent Nacylation of UDP-3-O-(acyl)-α-D-glucosamine, and is a target for new antibiotic development. Here we report the 2.6 Å crystal structure of the Escherichia coli LpxD homotrimer (EcLpxD). As is the case in Chlamydia trachomatis LpxD (CtLxpD), each EcLpxD chain consists of an N-terminal uridine-binding region, a left-handed parallel β-helix (LβH), and a C-terminal α-helical domain. The backbones of the LβH domains of the two enzymes are similar, as are the positions of key active site residues. The N-terminal nucleotide binding domains are oriented differently relative to the LβH regions, but are similar when overlaid on each other. The orientation of the EcLpxD tripeptide (residues 303-305), connecting the distal end of the LβH and the proximal end of the C-terminal helical domains, differs from its counterpart in CtLpxD (residues 311-312); this results in a 120°r otation of the C-terminal domain relative to the LβH region in EcLpxD versus CtLpxD. M290 of EcLpxD appears to cap the distal end of a hydrophobic cleft that binds the acyl chain of the R-3-hydroxyacyl-ACP donor substrate. Under standard assay conditions, wild-type EcLpxD prefers R,S-3-hydroxymyristoyl-ACP over R,S-3-hydroxypalmitoyl-ACP by a factor of 3, whereas the M290A mutant has the opposite selectivity. Both wild-type and M290A EcLpxD rescue the conditional lethality of E. coli RL25, a temperature-sensitive strain harboring point mutations in lpxD. Complementation with wild-type EcLpxD restores normal lipid A containing only N-linked hydroxymyristate to RL25 at 42 °C, as judged by mass spectrometry, whereas the M290A mutant generates multiple lipid A species containing one or two longer hydroxy fatty acids in place of the usual R-3-hydroxymyristate at positions 2 and 2′.LpxD catalyzes the third step of lipid A biosynthesis, the acyl-ACP dependent N-acylation of the intermediate UDP-3-O-(acyl)-α-D-glucosamine (Scheme 1) (1-4). LpxD is conserved among all Gram-negative bacteria that make lipid A and is required for growth (1-4). It is therefore an excellent target for the development of new antibiotics. Three related crystal structures of LpxD from Chlamydia trachomatis (CtLpxD) 1 have recently been reported by Buetow et al. (5): an "apo" structure at 2.7 Å resolution, and two structures with bound UDP-N-acetylglucosamine (UDP-GlcNAc) at 2.2 Å and 3.1 Å resolution (termed Complex I and Complex II, respectively) (5). These structures revealed that CtLpxD contains three nearly identical protein chains within its asymmetric unit (5), which coincide with the three subunits *Author to whom correspondence should be addressed: C. R. H. Raetz at (919) 684-3384; Fax (919) 684-8885; raetz@biochem.duke.edu. Supporting Information Available: A supplementary figure showing the amino acid sequence alignments of EcLpxD and CtLpxD is provided, along with a table showing the differences in the lengths of the connecting loops between the secondary structural elements of t...

At groundwater sites contaminated with chlorinated ethenes, fermentable substrates are often added to promote reductive dehalogenation by indigenous or augmented microorganisms. Contemporary bioremediation performance monitoring relies on nucleic acid biomarkers of key organohalide-respiring bacteria, such as Dehalococcoides mccartyi (Dhc). Metagenome sequencing of the commercial, Dhccontaining consortium, SDC-9, identified 12 reductive dehalogenase (RDase) genes, including pceA (two copies), vcrA, and tceA, and allowed for specific detection and quantification of RDase peptides using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Shotgun (i.e., untargeted) proteomics applied to the SDC-9 consortium grown with tetrachloroethene (PCE) and lactate identified 143 RDase peptides, and 36 distinct peptides that covered greater than 99% of the protein-coding sequences of the PceA, TceA, and VcrA RDases. Quantification of RDase peptides using multiple reaction monitoring (MRM) assays with 13 C-/ 15 N-labeled peptides determined 1.8 × 10 3 TceA and 1.2 × 10 2 VcrA RDase molecules per Dhc cell. The MRM mass spectrometry approach allowed for sensitive detection and accurate quantification of relevant Dhc RDases and has potential utility in bioremediation monitoring regimes.