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Comparative Study
. 2005 Aug;187(15):5292-300.
doi: 10.1128/JB.187.15.5292-5300.2005.

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

Affiliations
Comparative Study

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

Fiona McArthur et al. J Bacteriol. 2005 Aug.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Biosynthesis pathway of ADP-l-glycero-d-manno-heptose. The protein designations are indicated in the boxes. HldA and HldC are reserved only for those cases where the enzymes are individually encoded by independent genes (30).
FIG. 2.
FIG. 2.
Analysis of lipid A-core from parental strain and hldE mutants. The thick lines indicate the background E. coli strain used to transform recombinant plasmids encoding (as GST-fused proteins) full-length HldE (pFM1), HldE2 (ADP-heptose transferase domain; pFM2), HldE1 (heptokinase domain; pFM3), and the various mutant forms of HldE1. The background strains were SØ874 (wild type) (panels A, B, and C), FAM2 (SØ874 ΔhldE) (panel A), FAM4 (SØ874 ΔhldE2::Km) (panel B), and FAM3 (SØ874 ΔhldE1) (panel C).
FIG. 3.
FIG. 3.
Analysis of polypeptides by SDS-PAGE. Polypeptides corresponding to full-length HldE (pFM1), HldE2 (ADP-heptose transferase domain; pFM2), HldE1 (heptokinase domain; pFM3), the various mutant forms of HldE, and the GST protein fusion partner (pGEX-2T) are indicated by the arrows. All of the recombinant proteins were detected as GST-fused polypeptides. M, molecular weight markers in thousands.
FIG. 4.
FIG. 4.
Cluster tree of various members of the ribokinase superfamily with known crystal structures.
FIG. 5.
FIG. 5.
Alignment of E. coli ribokinase and HldE1 amino acid sequences. Only residues indicated in black type were used for the analysis. Gray shading indicate the conserved residues. Mutated amino acids in ATP binding and catalytic activity sites are indicated with boxes. The boxes above the alignment indicate the amino acids involved in forming the “lid” region.
FIG. 6.
FIG. 6.
Homology model of the E. coli HldE1 domain (heptokinase) based on the crystal structure of ribokinase (PDB entry 1RKA), using MODELLER. Alignment of HldE1 and ribokinase was performed using CLUSTAL W. The first 11 residues of HldE1 and the first 3 residues of the ribokinase were removed. The last 18 residues in HldE1 are not included in this model.
FIG. 7.
FIG. 7.
A, circular dichroism spectrum of HldE1, D264N, and E198D mutant proteins. B, spectrofluorometric profile based on the excitation of the HldE tryptophan 25 residue at 295 nm. The excitation slit was set to 1 nm; the emission slit was set to 2 nm. Maximum fluorescence for parental and mutant proteins showed maximum excitation at 338 ± 3 nm.
FIG. 8.
FIG. 8.
Size exclusion chromatography profiles obtained with wild-type HldE1, HldE1D264E, and HldE1N198E. The x axis corresponds to milliliters passed over the column, and the y axis indicates the absorption read in the detector at 280 nm. The height of the peak depends on the relative concentration of protein loaded into the column. Standards used to calibrate the column were blue dextran (2000 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). The predominant peak of elution (14.1 ml) in all cases corresponded to an approximate mass of 83 kDa, which is consistent with expected mass of the dimeric form of HldE1.
FIG. 9.
FIG. 9.
Western blot from a copurification experiment using a strain coexpressing a full-length HldE N terminally fused to GST and an HldE1 domain C terminally fused to the FLAG epitope tag. A cell-free lysate was passed through a GSTrap column to immobilize the GST-tagged protein. After washing with 50 times the column volume, proteins were eluted with reduced glutathione. The various fractions were examined by immunoblotting probed with antibodies against both GST and FLAG tags. The blots were visualized by fluorescence scanning with an Odyssey infrared imaging system (LI-COR Biosciences) using both 700-nm (for detection of the FLAG epitope) and 800-nm (for detection of the GST tag) channels (for more details, see Materials and Methods).

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