3FYO

Crystal structure of the triple mutant (N23C/D247E/P249A) of 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) from Neisseria meningitidis

Summary for 3FYO

• Entry DOI: 10.2210/pdb3fyo/pdb
• Related: 2qkf
• Descriptor: 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase, CHLORIDE ION, SODIUM ION, ... (6 entities in total)
• Functional Keywords: manno-octulosonate, synthase, lipopolysaccharide, kdop, kdo8 kdops, kdo8ps, tim barrel, biosynthesis, transferase, lyase, lipopolysaccharide biosynthesis
• Biological source: Neisseria meningitidis serogroup B
• Cellular location: Cytoplasm (By similarity): Q9JZ55
• Total number of polymer chains: 4
• Total formula weight: 122302.29

Authors

Jameson, G.B.,Parker, E.J.,Cochrane, F.P.,Patchett, M.L. (deposition date: 2009-01-22, release date: 2009-03-24, Last modification date: 2024-11-06)

Primary citation

Cochrane, F.C.,Cookson, T.V.,Jameson, G.B.,Parker, E.J.
Reversing evolution: re-establishing obligate metal ion dependence in a metal-independent KDO8P synthase
J.Mol.Biol., 390:646-661, 2009

PubMed Abstract: Two distinct groups of 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS), a key enzyme of cell-wall biosynthesis, differ by their requirement for a divalent metal ion for enzymatic activity. The unique difference between these groups is the replacement of the metal-binding Cys by Asn. Substitution of just this Asn for a Cys in metal-independent KDO8PS does not create the obligate metal-ion dependency of natural metal-dependent enzymes. We describe how three or four mutations of the metal-independent KDO8PS from Neisseria meningitidis produce a fully functional, obligately metal-dependent KDO8PS. For the substitutions Asn23Cys, Asp247Glu (this Asp binds to the metal ion in all metal-dependent KDO8PS) and Pro249Ala, and for double and triple combinations, mutant enzymes that contained Cys in place of Asn showed an increase in activity in the presence of divalent metal ions. However, combining these mutations with substitution by Ser of the Cys residue in the conserved (246)CysAspGlyPro(249) motif of metal-independent KDO8PS created enzymes with obligate metal dependency. The quadruple mutant (Asn23Cys/Cys246Ser/Asp247Glu/Pro249Ala) showed comparable activity to wild-type enzymes only in the presence of metal ions, with maximum activity with Cd(2+), the metal ion that is strongly inhibitory at micromolar concentrations for the wild-type enzyme. In the absence of metal ions, activity was barely detectable for this quadruple mutant or for triple mutants bearing both Cys246Ser and Asn23Cys mutations. The structures of NmeKDO8PS and its Asn23Cys/Asp247Glu/Pro249Ala and quadruple mutants at pH 4.6 were characterized at resolutions better than 1.85 A. Aged crystals of the Asn23Cys/Asp247Glu/Pro249Ala mutant featured a Cys23-Cys246 disulfide linkage, explaining the spectral bleaching observed when this mutant was incubated with Cu(2+). Such bleaching was not observed for the quadruple mutant. Reverse evolution to a fully functional obligately metal-dependent KDO8PS has been achieved with just three directed mutations for enzymes that have, at best, 47% identity between metal-dependent and metal-independent pairs.

PubMed: 19447118
DOI: 10.1016/j.jmb.2009.05.014

Experimental method

X-RAY DIFFRACTION (1.9 Å)