Kinetic characterization and quaternary structure of glutamate racemase from the periodontal anaerobe Fusobacterium nucleatum

Cofactor-independent glutamate racemases (GRs) that supply the d-glutamate required for biosynthesis of the peptidoglycan that encapsulates bacterial cells are attractive targets for the development of antibacterial drugs. Recombinant GR from Fusobacterium nucleatum (FnGR), a Gram-negative anaerobe involved in periodontal disease, was overproduced, purified, and characterized. Unlike most other GRs, FnGR is a pseudosymmetric enzyme, catalyzing the racemization of glutamate enantiomers with similar kinetic parameters ( k cat l → d = 17.4 ± 0.8 s - 1 , K m l → d = 1.04 ± 0.07 mM, k cat d → l = 26 ± 1 s - 1 , and K m d → l = 17.0 ± 0.1 mM ; pH optimum ∼8.5). Mutational analysis of residue 151 (A151V) located at the entryway to the active site revealed that FnGR is very sensitive to increased steric bulk at this position. Blue native-polyacrylamide gel electrophoresis, Ferguson plot analyses, and cross-linking studies, indicated that FnGR existed predominately as dimers. Unlike Bacillus subtilis GR, the presence of glutamate did not significantly alter the position of the monomer–dimer equilibrium of FnGR.