Mechanistic Insights into the Function of the α‐Helical Tail in Haemophilus influenzae 3‐Deoxy‐D‐manno‐Octulosonate 8‐Phosphate Phosphatase

Abstract

The eight carbon sugar, 3‐deoxy‐d‐manno‐octulosonate (KDO), serves as a fundamental link between lipid A and core oligosaccharides in the outer membrane of the Gram‐negative bacterial cell wall. Inhibition of KDO biosynthesis is lethal to most bacteria, implicating this pathway as an ideal target for novel antimicrobials. KdsC, which converts KDO 8‐phosphate to KDO, is one of four key enzymes in this pathway. In Haemophilus influenzae, KdsC is a homotetrameric protein with an α‐helical C‐terminal tail in each subunit. A truncated derivative of H. influenzae KdsC was constructed by cleavage of the last 18 C‐terminal amino acid residues through site‐directed mutagenesis to investigate the role of the α‐helical tail. Both native and truncated enzymes were cloned, overexpressed, and purified to apparent homogeneity. Preliminary data from crude 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, providing further insight into KdsC as a potential target to inhibit KDO biosynthesis. This work was funded by the National Science Foundation.