Mechanistic Insights into the Function of the α-Helical Tail in Haemophilus
Briski, Laurence A.
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The cell wall of Gram-negative bacteria is a double membrane structure which contains lipopolysaccharides (LPS) consisting of lipid A molecules, core oligosaccharides, and Oantigens in the outer membrane. The eight carbon sugar 3-deoxy-D-manno-octulosonate (KDO) is a highly conserved moiety in the LPS, serving as a fundamental link between lipid A and core oligosaccharide molecules. Inhibition of KDO biosynthesis is lethal to most bacteria, implicating this pathway as an ideal target for the development of novel antimicrobial agents. KDO 8-phosphate phosphatase (KdsC), which cleaves the phosphate group from KDO 8- phosphate to yield KDO, is one of four key enzymes in the KDO biosynthetic pathway. In Escherichia coli, KdsC has been characterized as a homotetrameric protein with each subunit containing a random-coiled C-terminal tail which has been shown to play a potential role in the enzyme’s catalytic cycle. In Haemophilus influenzae, KdsC similarly consists of a homotetrameric structure with an α-helical C-terminal tail in each subunit. A truncated (tail-less) derivative of H. influenzae KdsC was constructed by cleavage of the last eighteen C-terminal amino acid residues through site-directed mutagenesis to investigate the role of the α-helical tail. Both native and truncated H. influenzae KdsC were cloned, overexpressed, and purified to apparent homogeneity. Preliminary data from crude cell-free protein extracts showed that the activity of the native enzyme was 33-fold greater than that of the truncated derivative, as determined by a malachite green assay to assess inorganic phosphate release. These results provide further insight into KdsC as a potential target to inhibit KDO biosynthesis, and subsequently inhibit the proliferation of pathogenic Gram-negative bacteria.