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Biochemistry. 2013 Jul 9;52(27):4676-86. doi: 10.1021/bi400521f. Epub 2013 Jun 26.

Examining the role of intersubunit contacts in catalysis by 3-deoxy-d-manno-octulosonate 8-phosphate synthase.

Author information

1
Institute of Fundamental Sciences, Massey University , Palmerston North, New Zealand.

Abstract

3-Deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenolpyruvate and arabinose 5-phosphate (A5P) in the first committed step in the pathway to 3-deoxy-d-manno-octulosonate, a component in the cell wall of Gram-negative bacteria. KDO8PS is evolutionarily and structurally related to the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), which uses erythrose 4-phosphate in place of A5P. Both KDO8PS and type Iβ DAH7PS enzymes adopt similar homotetrameric associations with their active sites close to one of the interfaces. The conserved PAFLxR motif in KDO8PS and the corresponding GARNxQ motif in type Iβ DAH7PS, both on the short β4-α4 loop of the (β/α)8 barrel, form part of this interface and provide key contacts with substrates. This (112)PAFLxR(117) motif was mutated in Neisseria meningitidis KDO8PS in order to assess its role in enzyme function. Arg117 extends across the interface to provide guanidinium functionality in the A5P binding site of the adjacent subunit. Substitution Arg117Ala severely hampered catalysis, whereas substitution to Lys was tolerated better. Mutation of Phe114 to either Arg or Ala results in active proteins, but with substantially elevated Km(A5P) values. Mutant proteins that combine substitutions in this motif demonstrate poor catalytic function, and, although these mutated residues now structurally resemble their counterparts in the GARNxQ motif of type Iβ DAH7PS, no DAH7PS-like activity was observed. Analysis of the structures reveals that small changes in relative orientation of the subunits are important for the differences in active-site construction. Quaternary structure is therefore tightly linked to substrate specificity.

PMID:
23746359
DOI:
10.1021/bi400521f
[Indexed for MEDLINE]

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