Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis

Proc Natl Acad Sci U S A. 2012 Apr 17;109(16):6253-8. doi: 10.1073/pnas.1119894109. Epub 2012 Apr 2.

Abstract

WaaA is a key enzyme in the biosynthesis of LPS, a critical component of the outer envelope of Gram-negative bacteria. Embedded in the cytoplasmic face of the inner membrane, WaaA catalyzes the transfer of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to the lipid A precursor of LPS. Here we present crystal structures of the free and CMP-bound forms of WaaA from Aquifex aeolicus, an ancient Gram-negative hyperthermophile. These structures reveal details of the CMP-binding site and implicate a unique sequence motif (GGS/TX(5)GXNXLE) in Kdo binding. In addition, a cluster of highly conserved amino acid residues was identified which represents the potential membrane-attachment and acceptor-substrate binding site of WaaA. A series of site-directed mutagenesis experiments revealed critical roles for glycine 30 and glutamate 31 in Kdo transfer. Our results provide the structural basis of a critical reaction in LPS biosynthesis and allowed the development of a detailed model of the catalytic mechanism of WaaA.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites / genetics
  • Biocatalysis
  • Crystallography, X-Ray
  • Glutamic Acid / chemistry
  • Glutamic Acid / genetics
  • Glutamic Acid / metabolism
  • Glycine / chemistry
  • Glycine / genetics
  • Glycine / metabolism
  • Glycosyltransferases / chemistry*
  • Glycosyltransferases / genetics
  • Glycosyltransferases / metabolism
  • Gram-Negative Bacteria / enzymology
  • Gram-Negative Bacteria / genetics
  • Gram-Negative Bacteria / metabolism
  • Lipid A / biosynthesis
  • Lipopolysaccharides / biosynthesis*
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Interaction Domains and Motifs
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • Spectrometry, Fluorescence
  • Transferases / chemistry*
  • Transferases / genetics
  • Transferases / metabolism

Substances

  • Bacterial Proteins
  • Lipid A
  • Lipopolysaccharides
  • Membrane Proteins
  • Glutamic Acid
  • Transferases
  • Glycosyltransferases
  • 3-deoxy-D-manno-octulosonate transferase
  • Glycine