Abstract
The membrane-bound tetraacyldisaccharide-1-phosphate 4'-kinase, LpxK, catalyzes the sixth step of the lipid A (Raetz) biosynthetic pathway and is a viable antibiotic target against emerging Gram-negative pathogens. We report the crystal structure of lipid IVA, the LpxK product, bound to the enzyme, providing a rare glimpse into interfacial catalysis and the surface scanning strategy by which many poorly understood lipid modification enzymes operate. Unlike the few previously structurally characterized proteins that bind lipid A or its precursors, LpxK binds almost exclusively to the glucosamine/phosphate moieties of the lipid molecule. Steady-state kinetic analysis of multiple point mutants of the lipid-binding pocket pinpoints critical residues involved in substrate binding, and characterization of N-terminal helix truncation mutants uncovers the role of this substructure as a hydrophobic membrane anchor. These studies make critical contributions to the limited knowledge surrounding membrane-bound enzymes that act upon lipid substrates and provide a structural template for designing small molecule inhibitors targeting this essential kinase.
Highlights
LpxK is an essential membrane-bound kinase in the lipid A biosynthetic pathway
On identical plates that had been incubated at 42 °C, TG1/pTAG1 cells transformed with pBAD30 containing either E. coli lpxK or A. aeolicus lpxK were able to grow, whereas the empty vector could not (Fig. 2), indicating that A. aeolicus LpxK can perform the function of E. coli LpxK sufficiently well to complement a chromosomal knockout of the gene
Colony PCR confirmed the presence of the lpxK::kan insertion at the correct location on the chromosome. These results indicate that the molecular differences between E. coli and A. aeolicus DSMP do not prevent A. aeolicus LpxK from utilizing the E. coli-derived substrate and validate our pursuit of structural and kinetic studies involving A. aeolicus LpxK with the more accessible E. coli-derived reaction product lipid IVA
Summary
LpxK is an essential membrane-bound kinase in the lipid A biosynthetic pathway. Results: Structural and kinetic studies reveal the molecular basis of lipid binding. Steady-state kinetic analysis of multiple point mutants of the lipid-binding pocket pinpoints critical residues involved in substrate binding, and characterization of N-terminal helix truncation mutants uncovers the role of this substructure as a hydrophobic membrane anchor These studies make critical contributions to the limited knowledge surrounding membrane-bound enzymes that act upon lipid substrates and provide a structural template for designing small molecule inhibitors targeting this essential kinase. Knowledge of how LpxK interacts with the membrane and its lipid substrate will directly facilitate the development of small molecule antibiotics targeting this essential lipid A enzyme in Gram-negative bacteria and provide molecular insight into the activity of membrane-bound lipid modification enzymes, about which there exist precious little structural or kinetic data to date. Based on the directionality of the nucleotide phosphates in the AMP-PCP-
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
