期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 109, 期 32, 页码 12956-12961出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1206072109
关键词
lipid metabolism; endotoxin; integral monotopic protein; Walker motif
资金
- US Department of Energy, Office of Science, and the Office of Basic Energy Sciences [W-31-109-Eng-38]
- National Institutes of Health Grant [GM-51310]
In Gram-negative bacteria, the hydrophobic anchor of the outer membrane lipopolysaccharide is lipid A, a saccharolipid that plays key roles in both viability and pathogenicity of these organisms. The tetraacyldisaccharide 4'-kinase (LpxK) of the diverse P-loop-containing nucleoside triphosphate hydrolase superfamily catalyzes the sixth step in the biosynthetic pathway of lipid A, and is the only known P-loop kinase to act upon a lipid substrate at the membrane. Here, we report the crystal structures of apo- and ADP/Mg2+- bound forms of Aquifex aeolicus LpxK to a resolution of 1.9 angstrom and 2.2 angstrom, respectively. LpxK consists of two alpha/beta/alpha sandwich domains connected by a two-stranded beta-sheet linker. The N-terminal domain, which has most structural homology to other family members, is responsible for catalysis at the P-loop and positioning of the disaccharide-1-phosphate substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain, a substructure unique to LpxK, helps bind the nucleotide substrate and Mg2+ cation using a 25 degrees hinge motion about its base. Activity was severely reduced in alanine point mutants of conserved residues D138 and D139, which are not directly involved in ADP or Mg2+ binding in our structures, indicating possible roles in phosphoryl acceptor positioning or catalysis. Combined structural and kinetic studies have led to an increased understanding of the enzymatic mechanism of LpxK and provided the framework for structure-based antimicrobial design.
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