Abstract
3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3-deoxy-D-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 A (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (beta/alpha)(8) barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 A apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy.
Highlights
3-Deoxy-D-manno-octulosonate (KDO)1 is an 8-carbon sugar present in the lipopolysaccharide (LPS) of all Gram-negative bacteria [1]
Overall Structure—The asymmetric unit of the crystal contains a homotetramer of KDO8P synthase with 222 local symmetry (Fig. 2); each monomer has the fold of a typical (/␣)8 barrel as observed in the structure of triose-phosphate isomerase (TIM) [26]
The secondary structure elements of the E. coli KDO8P synthase (284 residues) and TIM from various sources (247–250 residues) superimpose well with the main differences occurring at the N terminus, where KDO8P synthase has an additional -hairpin that seals the N-terminal end of the barrel, and at the C terminus, where helix H8 of KDO8P synthase has a random coil extension (Fig. 2)
Summary
Protein Purification and Crystallization—The E. coli KDO8P synthase (KDO8PS) is encoded by the kdsA gene [12]. Ten ml from a small scale growth of the overexpressing strain were transferred to two flasks each containing 1 liter of Luria-Bertani medium supplemented with 100 g mlϪ1 ampicillin, and the culture was maintained under shaking at 37 °C until mid-log phase At this point, expression of KDO8P synthase was induced with 60 M isopropyl--D-thiogalactopyranoside, and the cells were grown for 4 h. Fractions containing active KDO8P synthase were pooled, and solid ammonium sulfate was added to a final concentration of 0.5 M. The structure determination of the enzyme employed the anomalous diffraction at multiple wavelengths (MAD) of a mercury derivative (Table I) obtained by soaking crystals for 24 h in a holding solution containing 9 mM mercurochrome (2Ј,7Ј-dibromo-4Ј-[hydroxymercurio]-fluorescein) and sodium sulfate in place of ammonium sulfate. Structure superposition and structure-based sequence alignment were carried out using the program LSQMAN [25]
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