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Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4'-kinase LpxK involved in lipid A biosynthesis.

Publication ,  Journal Article
Emptage, RP; Pemble, CW; York, JD; Raetz, CRH; Zhou, P
Published in: Biochemistry
April 2, 2013

The sixth step in the lipid A biosynthetic pathway involves phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) intermediate by the cytosol-facing inner membrane kinase LpxK, a member of the P-loop-containing nucleoside triphosphate (NTP) hydrolase superfamily. We report the kinetic characterization of LpxK from Aquifex aeolicus and the crystal structures of LpxK in complex with ATP in a precatalytic binding state, the ATP analogue AMP-PCP in the closed catalytically competent conformation, and a chloride anion revealing an inhibitory conformation of the nucleotide-binding P-loop. We demonstrate that LpxK activity in vitro requires the presence of a detergent micelle and formation of a ternary LpxK-ATP/Mg(2+)-DSMP complex. Using steady-state kinetics, we have identified crucial active site residues, leading to the proposal that the interaction of D99 with H261 acts to increase the pKa of the imidazole moiety, which in turn serves as the catalytic base to deprotonate the 4'-hydroxyl of the DSMP substrate. The fact that an analogous mechanism has not yet been observed for other P-loop kinases highlights LpxK as a distinct member of the P-loop kinase family, a notion that is also reflected through its localization at the membrane, lipid substrate, and overall structure.

Duke Scholars

Published In

Biochemistry

DOI

EISSN

1520-4995

Publication Date

April 2, 2013

Volume

52

Issue

13

Start / End Page

2280 / 2290

Location

United States

Related Subject Headings

  • Protein Conformation
  • Point Mutation
  • Phosphotransferases (Alcohol Group Acceptor)
  • Phosphates
  • Models, Molecular
  • Magnesium
  • Lipid A
  • Kinetics
  • Detergents
  • Crystallography, X-Ray
 

Citation

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Emptage, R. P., Pemble, C. W., York, J. D., Raetz, C. R. H., & Zhou, P. (2013). Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4'-kinase LpxK involved in lipid A biosynthesis. Biochemistry, 52(13), 2280–2290. https://doi.org/10.1021/bi400097z
Emptage, Ryan P., Charles W. Pemble, John D. York, Christian R. H. Raetz, and Pei Zhou. “Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4'-kinase LpxK involved in lipid A biosynthesis.Biochemistry 52, no. 13 (April 2, 2013): 2280–90. https://doi.org/10.1021/bi400097z.
Emptage RP, Pemble CW, York JD, Raetz CRH, Zhou P. Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4'-kinase LpxK involved in lipid A biosynthesis. Biochemistry. 2013 Apr 2;52(13):2280–90.
Emptage, Ryan P., et al. “Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4'-kinase LpxK involved in lipid A biosynthesis.Biochemistry, vol. 52, no. 13, Apr. 2013, pp. 2280–90. Pubmed, doi:10.1021/bi400097z.
Emptage RP, Pemble CW, York JD, Raetz CRH, Zhou P. Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4'-kinase LpxK involved in lipid A biosynthesis. Biochemistry. 2013 Apr 2;52(13):2280–2290.
Journal cover image

Published In

Biochemistry

DOI

EISSN

1520-4995

Publication Date

April 2, 2013

Volume

52

Issue

13

Start / End Page

2280 / 2290

Location

United States

Related Subject Headings

  • Protein Conformation
  • Point Mutation
  • Phosphotransferases (Alcohol Group Acceptor)
  • Phosphates
  • Models, Molecular
  • Magnesium
  • Lipid A
  • Kinetics
  • Detergents
  • Crystallography, X-Ray