Functional analysis of the glycero-manno-heptose 7-phosphate kinase domain from the bifunctional HldE protein, which is involved in ADP-L-glycero-D-manno-heptose biosynthesis

The core oligosaccharide component of the lipopolysaccharide can be subdivided into inner and outer core regions. In Escherichia coli, the inner core consists of two 3-deoxy-D-manno-octulosonic acid and three glycero-manno-heptose residues. The HIdE protein participates in the biosynthesis of ADP-glycero-manno-heptose precursors used in the assembly of the inner core. HIdE comprises two functional domains: an N-terminal region with homology to the ribokinase superfamily (HIdE1 domain) and a C-terminal region with homology to the cytidylyltransferase superfamily (HIdE2 domain). We have employed the structure of the E. coli ribokinase as a template to model the HIdE1 domain and predict critical amino acids required for enzyme activity. Mutation of these residues renders the protein inactive as determined in vivo by functional complementation analysis. However, these mutations did not affect the secondary or tertiary structure of purified HIdE1, as judged by fluorescence spectroscopy and circular dichroism. Furthermore, in vivo coexpression of wild-type, chromosomally encoded HIdE and mutant HldE1 proteins with amino acid substitutions in the predicted ATP binding site caused a dominant negative phenotype as revealed by increased bacterial sensitivity to novobiocin. Copurification experiments demonstrated that HIdE and HIdE1 form a complex in vivo. Gel filtration chromatography resulted in the detection of a dimer as the predominant form of the native HIdE1 protein. Altogether, our data support the notions that the HIdE functional unit is a dimer and that structural components present in each HIdE1 monomer are required for enzymatic activity.