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Literature summary for 5.1.1.3 extracted from

  • Dean, S.F.; Whalen, K.L.; Spies, M.A.
    Biosynthesis of a novel glutamate racemase containing a site-specific 7-hydroxycoumarin amino acid enzyme-ligand promiscuity revealed at the atomistic level (2015), ACS Cent. Sci., 1, 364-373 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 5.1.1.3

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3). The expression of both the plasmid pET15b encoding the enzyme and plasmid pEB-JYRS, encoding the non-natural tRNA harboring the L-(7-hydroxycoumarin-4-yl) ethylglycine (7HC) by an orthogonal tRNA/aminoacyl-tRNA synthetase pair and corresponding tRNA synthetase, within strain BL21(DE3) appears to reduce the overall fitness of the strain, as demonstrated by a reduced growth rate in liquid culture Bacillus subtilis

Protein Variants

Protein Variants Comment Organism
additional information Tyr53 mutated to the L-(7-hydroxycoumarin-4-yl) ethylglycine (7HC) functional group by site-directed mutagenesis provides ligand-associated fluorescent sensitivity to changes in the local environment (and thus serve as an allosteric reporter), without sacrificing particular contacts with ligands. The placement of the 7HC moiety at the surface of the enzyme, mutant GRY53/7HC mutant, remote from any ligand pockets, places it in a microenvironment where dielectric values are significantly larger, which restricts fluorescence changes to largely water polarization effects. The extraordinary sensitivity of the 7HC moiety within GRY53/7HC, and its incorporation into the dynamic region, provides a valuable experimental probe, quickly identifying a more open and solvated form, which is umwanted for high quality complexation. The GRY53/7HC mutant maintains the same KM value as the wild-type enzyme, but exhibits a 40fold decreased kcat Bacillus subtilis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.25
-
 L-glutamate wild-type enzyme, pH and temperature not specified in the publication Bacillus subtilis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-glutamate Bacillus subtilis
-
 D-glutamate
-
 r
L-glutamate Bacillus subtilis 168
-
 D-glutamate
-
 r

Organism

Organism UniProt Comment Textmining
Bacillus subtilis P94556
-
-
Bacillus subtilis 168 P94556
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-glutamate
-
 Bacillus subtilis D-glutamate
-
 r
L-glutamate
-
 Bacillus subtilis 168 D-glutamate
-
 r

Synonyms

Synonyms Comment Organism
RACE
-
 Bacillus subtilis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.3
-
 L-glutamate wild-type enzyme, pH and temperature not specified in the publication Bacillus subtilis

General Information

General Information Comment Organism
additional information molecular dynamics simulations on the enzyme have suggested particular regions that undergo relatively large changes, both in terms of substrate unbinding, as well as equilibrated enzyme-ligand complexes that show movement relative to one another (i.e., enzyme complexes with different types of active site small molecules equilibrated to distinct conformers) Bacillus subtilis
physiological function glutamate racemase (GR) catalyzes the cofactor independent stereoinversion of L- to D-glutamate for biosynthesis of bacterial cell walls. Glutamate racemase is a flexible enzyme capable of binding a variety of small molecules both in the active site and at allosteric binding pockets Bacillus subtilis