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 Å (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (␤/␣) 8 barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 Å 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. 3-Deoxy-D-manno-octulosonate (KDO)1 is an 8-carbon sugar present in the lipopolysaccharide (LPS) of all Gram-negative bacteria (1). KDO provides a link between lipid A, the membrane embedded moiety of LPS, and the O-antigen, an elongated polysaccharide chain that protrudes from the bacterial outer membrane into the surrounding environment and determines the antigenic specificity of the cell. Although the composition of the O-antigen varies between species and also between strains, the inner core region containing KDO is fairly constant among all Gram-negative bacteria (2).3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8P synthase, EC 4.1.2.16) plays a key role in the biosynthesis of KDO. This enzyme catalyzes the aldol-type condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P (precursor to KDO) and inorganic phosphate ( Fig. 1) (3). Dephosphorylation of KDO8P to KDO and synthesis of CMP-KDO (from CTP and KDO) occur prior to insertion of the sugar into LPS (2). Strains of Salmonella have been isolated with mutations in KDO8P synthase that confer temperature-sensitive growth (4, 5). Such strains fail to synthesize KDO at the nonpermissive temperature, which leads to the inhibition of LPS biosynthesis and, as a consequence, to the arrest of cell growth. These studies indicate that KDO8P synthase provides an essential function for bacterial homeostasis.Earlier studies have determined that the reaction of KDO8P synthesis is a sequential process in which the binding of PEP precedes the binding of A5P and the release of inorganic phosphate precedes the release of KDO8P (6). The condensation step of the reaction is stereospecific, involving the additio...

An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector. The protein was overexpressed in Escherichia coli and purified to homogeneity. Recombinant A. thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa. Unlike previously studied bacterial KDOPSs, which are tetrameric, A. thaliana KDOPS appears to be a dimer in solution. The optimum temperature of the enzyme is 65 degrees C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity. The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme. Kinetic studies showed that the enzyme follows a sequential mechanism with K(m)=3.6 microM for phosphoenolpyruvate and 3.8 microM for D-arabinose 5-phosphate and kcat=5.9 s(-1) at 37 degrees C. On the basis of the characterization of A. thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs.

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase (EC 4.1. 2.16) (KDO 8-P synthase) that catalyzes the condensation of D-arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) and inorganic phosphate (Pi) was inactivated by the thiol-modifying reagents 5,5-dithiobis (2-nitrobenzoate) (DTNB) and methyl methanethiosulfonate (MMTS). Reaction of cloned native KDO 8-P synthase with DTNB correlated with modification of two of the four cysteine sulfhydryls per monomer of enzyme and total loss of enzymatic activity which could be partially restored by treatment with dithiothreitol (DTT). Cyanolysis of the DTNB-inactivated enzyme with KCN led to the elimination of 2 equiv of 5-thio-2-nitrobenzoate and partial recovery of activity. The presence of either substrate(s) or product(s) provided no protection against inactivation nor affected the number of cysteines modified, indicating that the cysteines modified are most likely not at the active site of KDO 8-P synthase. Titration of denatured enzyme with DTNB resulted in the modification of all four cysteines. After treatment of native enzyme with MMTS, no cysteines could be titrated with DTNB and no enzymatic activity could be detected. Treatment of the MMTS-inactivated KDO 8-P synthase with DTT resulted in restoration of enzymatic activity and the presence of two DTNB-titratable cysteine residues. Based on these observations and a report that KDO 8-P synthase is inactivated in a time-dependent manner with 3-bromopyruvate and that the substrate PEP protects against this inactivation, all four cysteines (38, 166, 206, and 249) were individually mutated to alanines via a modified PCR methodology. The C206A and C249A mutants were both enzymatically active with K(m) and Vmax values approximately identical to those of wild-type KDO 8-P synthase, and both native mutants reacted with DTNB to modify only one of the three remaining cysteine sulfhydryls per monomer of enzyme. Titration of denatured C206A and C249A mutants resulted in the modification of three cysteines. The C38A and C166A mutants were both for the most part enzymatically inactive. Titration of native C38A and C166A with DTNB resulted in modification of two cysteines while titration of the denatured mutant protein resulted in modification of the three remaining cysteines. Circular dichroism measurements of wild-type KDO 8-P synthase and the four C --> A mutants indicate modest but significant changes in the structure of the mutants. These results indicate that C206 and C249 in native KDO 8-P synthase are readily accessible to the modification reagent DTNB and therefore inactivation may result from structural changes in the DTNB-modified KDO 8-P synthase or blockage of access of substrates to the active site. The C38 and C166 in native KDO 8-P synthase are inaccessible to the modification reagent DTNB, indicating that they are located in the interior of KDO 8-P synthase, and loss of activity in the C38A and C166A mutants suggests their e...

3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the biosynthesis of an essential component of the lipopolysaccharide of all Gram-negative bacteria. The structure and mechanism of KDO8P synthase are being actively studied as this enzyme represents an important target for antibiotic therapy. The structure of the Escherichia coli KDO8P synthase in cubic crystals (space group I23) has recently been determined and the enzyme shown to be a tetramer of identical subunits. However, this information is challenged by biochemical studies, which suggest that the enzyme behaves in solution as a homotrimer. Here, the preparation and preliminary X-ray analysis of monoclinic crystals of KDO8P synthase are reported. The crystals belong to space group P2(1), with unit-cell parameters a approximately 50, b approximately 140, c approximately 74 A, beta approximately 105 degrees. The structure of KDO8P synthase in the monoclinic crystal form was determined by molecular replacement, using as a search model one of the subunits of the enzyme in the cubic crystals. A tetramer of KDO8P synthase with 222 local symmetry is also present in the asymmetric unit of the P2(1) crystals, with a solvent content of 43%. The observation that the same quaternary structure of KDO8P synthase is observed in two different crystal forms belonging to distinct crystal systems (monoclinic and cubic) suggests that a tetramer is the native form of the enzyme.

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