title: Specificity and mutational analysis of the metal-dependent 3-deoxy-D-manno-octulosonate 8-phosphate synthase from Acidithiobacillus ferrooxidans.
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 degrees 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.
authors.affiliation: Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, Christchurch, New Zealand.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Allison
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Yeoman
authors.forename: Richard D
authors.initials: RD
authors.lastname: Hutton
authors.forename: Fiona C
authors.initials: FC
authors.lastname: Cochrane
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Jameson
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Parker
mesh_terms: D042763:Acidithiobacillus / Q000201:enzymology*; D000446:Aldehyde-Lyases / Q000235:genetics* / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D007477:Ions; D008670:Metals; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases