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+/-0.8s(-1), K(m)(L-->D)=1.04+/-0.07mM, k(cat)(D-->L)=26+/-1s(-1), and K(m)(D-->L)=1.7+/-0.1mM; [corrected] pH optimum approximately 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.
Potrykus
Joanna
J
Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS, Canada.
Flemming
Janette
J
Bearne
Stephen L
SL
D000409:Alanine / Q000378:metabolism; D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D018441:Biofilms; D003001:Cloning, Molecular; D055808:Drug Discovery; D016967:Fusobacterium nucleatum / Q000201:enzymology* / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010510:Periodontal Diseases / Q000382:microbiology; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity
D000591:Amino Acid Isomerases; C062292:glutamate racemase; D000409:Alanine