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Literature summary for 5.1.1.3 extracted from

  • Potrykus, J.; Flemming, J.; Bearne, S.
    Kinetic characterization and quaternary structure of glutamate racemase from the periodontal anaerobe Fusobacterium nucleatum (2009), Arch. Biochem. Biophys., 491, 16-24.
    View publication on PubMed
Search for more literature for 5.1.1.3

Application

Application Comment Organism
drug development enzyme is an attractive target for the development of antibacterial agents Bacillus subtilis
drug development enzyme is an attractive target for the development of antibacterial agents Fusobacterium nucleatum subsp. nucleatum

Cloned(Commentary)

Cloned (Comment) Organism
overexpression in BL21(DE3) Escherichia coli as fusion protein bearing an N-terminal hexahistidine tag Bacillus subtilis
overexpression in BL21(DE3) Escherichia coli as fusion protein bearing an N-terminal hexahistidine tag Fusobacterium nucleatum subsp. nucleatum

Protein Variants

Protein Variants Comment Organism
A151V production by site-directed mutagenesis, alanine ist essential for activity, mutation of residue 151 located at the entryway to the active site reveals that FnGR is very sensitive to increased steric bulk at this position Fusobacterium nucleatum subsp. nucleatum
V149A while V149A BsGR exhibits a 3- and 6fold increase in the value of Km for L- and D-glutamate relative to wild-type BsGR, respectively, the values of kcat are slightly increased relative to the wild-type enzyme Bacillus subtilis

Inhibitors

Inhibitors Comment Organism Structure
additional information although Tris-HCl isthe buffer most commonly employed when assaying glutamate racemases from various microbial sources, FnGR is slightly inhibited in this buffer, kcat values are reduced 14% and 25% in the L-D and D-L reaction directions at pH 8.0, respectively relative to the corresponding values observed using the phosphate assay buffer Fusobacterium nucleatum subsp. nucleatum

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.9
-
 L-glutamate His-tagged recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
1.04
-
 L-glutamate recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
1.5
-
 D-glutamate His-tagged recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
1.7
-
 D-glutamate His-tagged recombinant BsGR, in potassium phosphate buffer (10 mM, pH 8.0) Bacillus subtilis
1.7
-
 D-glutamate recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
10
-
 D-glutamate His-tagged recombinant BsGR mutant V149A, in potassium phosphate buffer (10 mM, pH 8.0) Bacillus subtilis
14
-
 L-glutamate His-tagged recombinant BsGR, in potassium phosphate buffer (10 mM, pH 8.0) Bacillus subtilis
26
-
 L-2-aminoadipic acid His-tagged recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
47
-
 L-glutamate His-tagged recombinant BsGR mutant V149A, in potassium phosphate buffer (10 mM, pH 8.0) Bacillus subtilis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
32050
-
 calculated His-tagged FnGR Fusobacterium nucleatum subsp. nucleatum
32130
-
 calculated His-BsGR V149A Bacillus subtilis
32160
-
 calculated His-tagged BsGR Bacillus subtilis
34000
-
 BsGR, determined by gel filtration Bacillus subtilis
35000
-
 FnGR, determined by SDS-PAGE Fusobacterium nucleatum subsp. nucleatum

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
D-glutamate Fusobacterium nucleatum subsp. nucleatum enzyme catalyzes the formation of D-glutamate from L-glutamate through a 1,1-proton transfer mechanism which reversibly inverts the stereochemistry at the alpha-carbon of glutamate L-glutamate
-
 r
L-2-aminoadipic acid Bacillus subtilis
-
 D-2-aminoadipic acid
-
 ?
L-2-aminoadipic acid Fusobacterium nucleatum subsp. nucleatum
-
 D-2-aminoadipic acid
-
 ?
L-glutamate Bacillus subtilis enzyme catalyzes the formation of D-glutamate from L-glutamate through a 1,1-proton transfer mechanism which reversibly inverts the stereochemistry at the alpha-carbon of glutamate D-glutamate
-
 r
L-glutamate Fusobacterium nucleatum subsp. nucleatum enzyme catalyzes the formation of D-glutamate from L-glutamate through a 1,1-proton transfer mechanism which reversibly inverts the stereochemistry at the alpha-carbon of glutamate D-glutamate
-
 r

Organism

Organism UniProt Comment Textmining
Bacillus subtilis P94556
-
-
Fusobacterium nucleatum subsp. nucleatum Q8REE6
-
-

Purification (Commentary)

Purification (Comment) Organism
by using of nickel ion affinity chromatography at 4°C Bacillus subtilis
by using of nickel ion affinity chromatography at 4°C Fusobacterium nucleatum subsp. nucleatum

Storage Stability

Storage Stability Organism
no loss of activity is noted after the enzyme is stored at 80°C for more than 8 months, at least 80% of enzyme activity is retained after incubation for 8 h at 4°C Fusobacterium nucleatum subsp. nucleatum

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
D-glutamate enzyme catalyzes the formation of D-glutamate from L-glutamate through a 1,1-proton transfer mechanism which reversibly inverts the stereochemistry at the alpha-carbon of glutamate Fusobacterium nucleatum subsp. nucleatum L-glutamate
-
 r
L-2-aminoadipic acid
-
 Bacillus subtilis D-2-aminoadipic acid
-
 ?
L-2-aminoadipic acid
-
 Fusobacterium nucleatum subsp. nucleatum D-2-aminoadipic acid
-
 ?
L-glutamate enzyme catalyzes the formation of D-glutamate from L-glutamate through a 1,1-proton transfer mechanism which reversibly inverts the stereochemistry at the alpha-carbon of glutamate Bacillus subtilis D-glutamate
-
 r
L-glutamate enzyme catalyzes the formation of D-glutamate from L-glutamate through a 1,1-proton transfer mechanism which reversibly inverts the stereochemistry at the alpha-carbon of glutamate Fusobacterium nucleatum subsp. nucleatum D-glutamate
-
 r

Subunits

Subunits Comment Organism
dimer determined by blue native PAGE, FnGR is a pseudosymmetric enzyme, the presence of glutamate does not significantly alter the position of the monomer-dimer equilibrium of the enzyme Fusobacterium nucleatum subsp. nucleatum
monomer determined by gel filtration, BsGR is a monomeric enzyme, which dimerizes in the presence of either 10 mM D- or L-glutamate Bacillus subtilis

Synonyms

Synonyms Comment Organism
BsGR
-
 Bacillus subtilis
FnGR
-
 Fusobacterium nucleatum subsp. nucleatum
glutamate racemase
-
 Bacillus subtilis
glutamate racemase
-
 Fusobacterium nucleatum subsp. nucleatum

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.4
-
 L-2-aminoadipic acid His-tagged recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
8.3
-
 D-glutamate His-tagged recombinant BsGR, in potassium phosphate buffer (10 mM, pH 8.0) Bacillus subtilis
13.8
-
 L-glutamate His-tagged recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
15
-
 D-glutamate His-tagged recombinant BsGR mutant V149A, in potassium phosphate buffer (10 mM, pH 8.0) Bacillus subtilis
17.4
-
 L-glutamate recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
22
-
 D-glutamate His-tagged recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
26
-
 D-glutamate recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
63
-
 L-glutamate His-tagged recombinant BsGR, in potassium phosphate buffer (10 mM, pH 8.0) Bacillus subtilis
76
-
 L-glutamate His-tagged recombinant BsGR mutant V149A, in potassium phosphate buffer (10 mM, pH 8.0) Bacillus subtilis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.5
-
 5 mM Tris-HCl Bacillus subtilis
8.5
-
 5 mM Tris-HCl Fusobacterium nucleatum subsp. nucleatum

pH Range

pH Minimum pH Maximum Comment Organism
8 8.5 FnGR is active over a broad pH range, with the maximum of activity between pH 8.0 and 8.5 for the reaction in the L-D direction Fusobacterium nucleatum subsp. nucleatum
8.5 9 FnGR is active over a broad pH range, with the maximum of activity between pH 8.0 and 8.5 for the reaction in the D-L direction Fusobacterium nucleatum subsp. nucleatum

General Information

General Information Comment Organism
physiological function peptidoglycan synthesis Bacillus subtilis
physiological function peptidoglycan synthesis Fusobacterium nucleatum subsp. nucleatum

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.09
-
 L-2-aminoadipic acid His-tagged recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
1.5
-
 D-glutamate His-tagged recombinant BsGR mutant V149A, in potassium phosphate buffer (10 mM, pH 8.0) Bacillus subtilis
1.8
-
 L-glutamate His-tagged recombinant BsGR mutant V149A, in potassium phosphate buffer (10 mM, pH 8.0) Bacillus subtilis
4
-
 D-glutamate His-tagged recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
4.6
-
 L-glutamate His-tagged recombinant BsGR, in potassium phosphate buffer (10 mM, pH 8.0) Bacillus subtilis
5
-
 D-glutamate His-tagged recombinant BsGR, in potassium phosphate buffer (10 mM, pH 8.0) Bacillus subtilis
15
-
 L-glutamate His-tagged recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
15
-
 D-glutamate recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum
17
-
 L-glutamate recombinant FnGR, in potassium phosphate buffer (10 mM, pH 8.0) Fusobacterium nucleatum subsp. nucleatum