Changes in quaternary structure cause a kinetic asymmetry of glutamate racemase‐catalyzed homocysteic acid racemization

Abstract

Glutamate racemases (GR) catalyze the racemization of d- and l-glutamate and are targets for the development of antibiotics. We demonstrate that GR from the periodontal pathogen Fusobacterium nucleatum (FnGR) catalyzes the racemization of d-homocysteic acid (d-HCA), while l-HCA is a poor substrate. This enantioselectivity arises because l-HCA perturbs FnGR's monomer-dimer equilibrium toward inactive monomer. The inhibitory effect of l-HCA may be overcome by increasing the total FnGR concentration or by adding glutamate, but not by blocking access to the active site through site-directed mutagenesis, suggesting that l-HCA binds at an allosteric site. This phenomenon is also exhibited by GR from Bacillus subtilis, suggesting that enantiospecific, "substrate"-induced dissociation of oligomers to form inactive monomers may furnish a new inhibition strategy.

Upregulated Palmitoleate and Oleate Production in Escherichia coli Promotes Gentamicin Resistance

“…Moreover, the increase in glutamate in this study also indicated more biomaterials available for the synthesis of peptidoglycan in the cell wall of gentamicin-resistant E. coli [ 30 ]. The glutamate racemases catalyzing the synthesis of D-glutamate from its L-isomer could be used as targets for antibiotic treatment and the development of antibiotics [ 31 ].…”

Racemases and epimerases operating through a 1,1-proton transfer mechanism: reactivity, mechanism and inhibition

“…Typical examples include Fusobacterium nucleatum and B. subtilis glutamate racemase, which have K m values of 1.04 and 1.07 mM and 14 and 1.24 mM, respectively. 166 In contrast, O -ureidoserine racemase which has reported K m values 46 of 12 and 32 mM for S - and R-O -ureidoserine, respectively. In contrast, the K m for S , S -diaminopimelate (2,6-diaminoheptanedioic acid) for M. tuberculosis diaminopimelate epimerase is only 166 μM, 167 significantly lower than the K m values for other amino-acid racemases/epimerases.…”

“…Similarly, k cat /K m is reported to be 16 730 and 15 294 M À1 s À1 for F. nucleatum glutamate racemase for S-and R-Glu, while the corresponding values are 3000 and 3806 M À1 s À1 for the B. subtilis enzyme. 166 In contrast, M. tuberculosis diaminopimelate epimerase 167 k cat /K m values for other racemases and epimerases tend to be higher and this is often related to the lower K m values observed for these larger substrates. For example, mandelate racemase (6.2 and 6.5 Â 10 5 M À1 s À1 for S-and R-mandelate 91 ) and the M. tuberculosis homologue of AMACR (5.23 Â 10 6 and 6.0 Â 10 6 M À1 s À1 for S-and R-ibuprofenoyl-CoA, 146 respectively).…”

“…Similarly, k cat / K m is reported to be 16 730 and 15 294 M −1 s −1 for F. nucleatum glutamate racemase for S - and R -Glu, while the corresponding values are 3000 and 3806 M −1 s −1 for the B. subtilis enzyme. 166 In contrast, M. tuberculosis diaminopimelate epimerase 167 has a very modest k cat / K m of 883 M −1 s −1 . On the other hand, RacX 168 has extremely low k cat / K m values of 2.86 and 3.23 M −1 s −1 for S - and R -Lys, while YgeA 168 has k cat / K m values of 45.8 and 45.8 M −1 s −1 for S - and R -His.…”