A single point mutation in 3-deoxy-D-manno-octulosonate-8-phosphate synthase is responsible for temperature sensitivity in a mutant strain of Salmonella typhimurium.
Salmonella typhimurium mutants conditionally deficient in 3-deoxy-d-manno-octulosonate-8-phosphate (KDO8P) synthase activity play a central role in our understanding of lipopolysaccharide function in enteric bacteria. The detailed characterization of KDO8P synthase from such a mutant, however, has not been previously reported. To address this issue KDO8P synthase from S. typhimurium AG701 and from a related temperature-sensitive strain (S. typhimurium AG701i50) have been overexpressed in Escherichia coli and purified to homogeneity. The enzyme from the temperature-sensitive strain has a single proline to serine substitution at position 145, leading to an increase in K(m) for both substrates, d-arabinose 5-phosphate and phosphoenolpyruvate. Analytical gel filtration and native polyacrylamide gel electrophoresis indicate that this enzyme also has an altered oligomeric state. These observations are rationalized through an examination of the structure of E. coli KDO8P synthase, which has 93% sequence identity to the enzyme from S. typhimurium.
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
W P
WP
Taylor
G Y
GY
Sheflyan
R W
RW
Woodard
D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D007700:Kinetics; D008070:Lipopolysaccharides / Q000737:chemistry; D008958:Models, Molecular; D010428:Pentosephosphates / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D013696:Temperature
D008070:Lipopolysaccharides; D010428:Pentosephosphates; D011994:Recombinant Proteins; C028055:arabinose 5-phosphate; D010728:Phosphoenolpyruvate; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases