Specificity and mutational analysis of the metal-dependent 3-deoxy-d-manno-octulosonate 8-phosphate synthase from Acidithiobacillus ferrooxidans |
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Authors: | Timothy M. Allison Jeffrey A. Yeoman Richard D. Hutton Fiona C. Cochrane Geoffrey B. Jameson Emily J. Parker |
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Affiliation: | 1. Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand;2. Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, Christchurch, New Zealand |
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Abstract: | 3-Deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenol pyruvate and d-arabinose 5-phosphate to generate KDO8P. This reaction is part of the biosynthetic pathway to 3-deoxy-d-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Two distinct groups of KDO8PSs exist, differing by the absolute requirement of a divalent metal ion. In this study Acidithiobacillus ferrooxidans KDO8PS has been expressed and purified and shown to require a divalent metal ion, with Mn2+, Co2+ and Cd2+ (in decreasing order) being able to restore activity to metal-free enzyme. Cd2+ significantly enhanced the stability of the enzyme, raising the Tm by 14 °C. d-Glucose 6-phosphate and d-erythrose 4-phosphate were not substrates for A. ferrooxidans KDO8PS, whereas 2-deoxy-d-ribose 5-phosphate was a poor substrate and there was negligible activity with d-ribose 5-phosphate. The 243AspGlyPro245 motif is absolutely conserved in the metal-independent group of synthases, but the Gly and Pro sites are variable in the metal-dependent enzymes. Substitution of the putative metal-binding Asp243 to Ala in A. ferrooxidans KDO8PS gave inactive enzyme, whereas substitutions Asp243Glu or Pro245Ala produced active enzymes with altered metal-dependency profiles. Prior studies indicated that exchange of a metal-binding Cys for Asn converts metal-dependent KDO8P synthase into a metal-independent form. Unexpectedly, this mutation in A. ferrooxidans KDO8P synthase (Cys21Asn) gave inactive enzyme. This finding, together with modest activity towards 2-deoxy-d-ribose 5-phosphate suggests similarities between the A. ferrooxidans KDO8PS and the related metal-dependent 3-deoxy-d-arabino-heptulosonate phosphate synthase, and highlights the importance of the AspGlyPro loop in positioning the substrate for effective catalysis in all KDO8P synthases. |
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Keywords: | A5P, d-arabinose 5-phosphate Aae, Aquifex aeolicus BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane Apy, Aquifex pyrophilus DAH7P, 3-deoxy-d-arabino-heptulosonate 7-phosphate DSF, Differential scanning fluorimetry DTT, dithiothreitol E4P, d-erythrose 4-phosphate Eco, Escherichia coli EDTA, ethylenediaminetetraacetic acid IPTG, isopropyl β-d-thiogalactopyranoside ITC, isothermal titration calorimetry KDO, 3-deoxy-d-manno-octulosonate KDO8P, 3-deoxy-d-manno-octulosonate 8-phosphate KDO8PS, 3-deoxy-d-manno-octulosonate 8-phosphate synthase LPS, lipopolysaccharide MWCO, molecular weight cut-off Nme, Neisseria meningitidis PEP, phosphoenol pyruvate SEC, size-exclusion chromatography |
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