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 HldE protein participates in the biosynthesis of ADP-glycero-manno-heptose precursors used in the assembly of the inner core. HldE comprises two functional domains: an N-terminal region with homology to the ribokinase superfamily (HldE1 domain) and a C-terminal region with homology to the cytidylyltransferase superfamily (HldE2 domain). We have employed the structure of the E. coli ribokinase as a template to model the HldE1 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 HldE1, as judged by fluorescence spectroscopy and circular dichroism. Furthermore, in vivo coexpression of wild-type, chromosomally encoded HldE 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 HldE and HldE1 form a complex in vivo. Gel filtration chromatography resulted in the detection of a dimer as the predominant form of the native HldE1 protein. Altogether, our data support the notions that the HldE functional unit is a dimer and that structural components present in each HldE1 monomer are required for enzymatic activity.
McArthur
Fiona
F
Department of Microbiology and Immunology, Siebens Drake Research Institute, Schulich School of Medicine, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Andersson
C Evalena
CE
Loutet
Slade
S
Mowbray
Sherry L
SL
Valvano
Miguel A
MA
D000244:Adenosine Diphosphate / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D006539:Heptoses / Q000096:biosynthesis*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013698:Templates, Genetic
D006539:Heptoses; D009097:Multienzyme Complexes; C026312:glycero-manno-heptose; D000244:Adenosine Diphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C022534:ribokinase; D009713:Nucleotidyltransferases; C445302:D,D heptose 7-phosphate kinase-D,D-heptose 1-phosphate adenylyltransferase, E coli