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J. Biol. Chem., Vol. 275, Issue 41, 32141-32146, October 13, 2000

This Article Full Text Full Text (PDF) All Versions of this Article: 275/41/32141    most recent M001900200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Taylor, W. P. Articles by Woodard, R. W. Search for Related Content PubMed PubMed Citation Articles by Taylor, W. P. Articles by Woodard, R. W. Social Bookmarking                      What's this?

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^*

William P. Taylor, Galina Ya. Sheflyan, and Ronald W. Woodard

From the Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065

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.

The atomic coordinates and the structure factors (code 1D9E) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

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