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Information on EC 5.1.1.3 - glutamate racemase and Organism(s) Bacillus subtilis and UniProt Accession P94556
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5.1.1.3 glutamate racemaseIUBMB Comments
A pyridoxal-phosphate protein.
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Word Map
- 5.1.1.3
- peptidoglycan
- l-glutamate
-
cofactor-independent
- drug development
- pediococcus
-
poly-gamma-glutamate
- pyrophilus
- pentosaceus
- fermenti
-
ciceri
-
stereoinversion
- medicine
- synthesis
The taxonomic range for the selected organisms is: Bacillus subtilis
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
glutamate racemase, race2, race1, d-glutamate racemase, glutamic acid racemases, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
glutamate racemase
RACE
Racemase, glutamate
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
L-glutamate = D-glutamate
MurI is a member of the two-base mechanism (dual-cysteine) racemase family, where two essential active-site cysteine residues act as catalytic base and acid to stereospecifically de- and reprotonate, respectively, the alpha position of glutamate in order to enact substrate racemization. C185 of BsMurI corresponds to the essential catalytic cysteine residue that deprotonates an incoming L-glutamate substrate (or reprotonates a carbanionic intermediate to form the D-stereoconfiguration)
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
racemization
racemization
-
-
-
-
PATHWAY SOURCE
PATHWAYS
-
-, -
SYSTEMATIC NAME
IUBMB Comments
glutamate racemase
A pyridoxal-phosphate protein.
CAS REGISTRY NUMBER
COMMENTARY
9024-08-2
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
L-Glu
?
glutamate racemase is mainly concerned in D-Glu synthesis for poly-gamma-glutamate production
-
-
?
L-glutamate
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
-
-
r
D-glutamate
L-glutamate
-
catalytic action of glutamate racemase is driven by its own substrate, D-glutamate
-
-
r
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
D-glutamate
L-glutamate
-
catalytic action of glutamate racemase is driven by its own substrate, D-glutamate
-
-
r
L-Glu
?
glutamate racemase is mainly concerned in D-Glu synthesis for poly-gamma-glutamate production
-
-
?
L-glutamate
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
-
-
r
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
glutamate racemase (MurI) is a pyridoxal 5'-phosphate-independent racemase
-
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
beta-Chloro-D-alanine
BCDA, its primary target is glutamate racemase, poor activity oagainst alanine racemase activity, potent antituberculosis activity. BCDA does not inhibit the D-alanine pathway in intact cells, consistent with its poor in vitro activity, it is instead an irreversible mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of peptidoglycan biosynthesis. Inhibition kinetics, overview. BCDA-treated BsMurI has a single cysteine residue, C185, that is the sole site of modification in over 95% of the inactivated protein. BCDA inhibition of MurI is mechanism-based as opposed to arising from nonspecific interaction with suitably configured cysteine thiols
beta-chloro-L-alanine
BCLA, modifies BsMurI predominantly at C74, the cysteine residue responsible for deprotonation of an incoming D-Glu substrate
additional information
no inhibition by beta-fluoroalanine (BFA, racemic mixture) and O-acetyl-D-serine (OADS)
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
14
L-glutamate
wild type enzyme, 50 mM boric acid, 100 mM KCl, 0.2 mM dithiothreitol, pH 8.0 at 25°C in the presence of 0.22 microM glutamate racemase
1.7
D-glutamate
His-tagged recombinant BsGR, in potassium phosphate buffer (10 mM, pH 8.0)
10
D-glutamate
His-tagged recombinant BsGR mutant V149A, in potassium phosphate buffer (10 mM, pH 8.0)
0.25
L-glutamate
wild-type enzyme, pH and temperature not specified in the publication
14
L-glutamate
His-tagged recombinant BsGR, in potassium phosphate buffer (10 mM, pH 8.0)
47
L-glutamate
His-tagged recombinant BsGR mutant V149A, in potassium phosphate buffer (10 mM, pH 8.0)
0.25
D-glutamate
wild type enzyme, at 25 °C in 50 mM Tris-HCl (pH 8.0), 5 mM NAD+, 0.5 mM iodonitrotetrazolium chloride, 2.5 mM ADP, 20 units of L-glutamate dehydrogenase, 2 units of diaphorase and D-glutamate
8.6
D-glutamate
mutant T76A, at 25 °C in 50 mM Tris-HCl, pH 8.0, 5 mM NAD+, 0.5 mM iodonitrotetrazolium chloride, 2.5 mM ADP, 20 units of L-glutamate dehydrogenase, 2 units of diaphorase and D-glutamate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
8.3
D-glutamate
His-tagged recombinant BsGR, in potassium phosphate buffer (10 mM, pH 8.0)
15
D-glutamate
His-tagged recombinant BsGR mutant V149A, in potassium phosphate buffer (10 mM, pH 8.0)
1.3
L-glutamate
wild-type enzyme, pH and temperature not specified in the publication
63
L-glutamate
His-tagged recombinant BsGR, in potassium phosphate buffer (10 mM, pH 8.0)
76
L-glutamate
His-tagged recombinant BsGR mutant V149A, in potassium phosphate buffer (10 mM, pH 8.0)
0.43
D-glutamate
mutant T76A, at 25°C in 50 mM Tris-HCl (pH 8.0), 5 mM NAD+, 0.5 mM iodonitrotetrazolium chloride, 2.5 mM ADP, 20 units of L-glutamate dehydrogenase, 2 units of diaphorase and D-glutamate
1.3
D-glutamate
wild type enzyme, at 25°C in 50 mM Tris-HCl, pH 8.0, 5 mM NAD+, 0.5 mM iodonitrotetrazolium chloride, 2.5 mM ADP, 20 units of L-glutamate dehydrogenase, 2 units of diaphorase and D-glutamate
87
L-glutamate
wild type enzyme, 50 mM boric acid, 100 mM KCl, 0.2 mM dithiothreitol, pH 8.0, at 25°C in the presence of 0.22 microM glutamate racemase
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.5
D-glutamate
His-tagged recombinant BsGR mutant V149A, in potassium phosphate buffer (10 mM, pH 8.0)
5
D-glutamate
His-tagged recombinant BsGR, in potassium phosphate buffer (10 mM, pH 8.0)
1.8
L-glutamate
His-tagged recombinant BsGR mutant V149A, in potassium phosphate buffer (10 mM, pH 8.0)
4.6
L-glutamate
His-tagged recombinant BsGR, in potassium phosphate buffer (10 mM, pH 8.0)
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.042
2-hydroxy-3,4,5-trioxocyclopent-1-en-1-olate
-
pH and temperature not specified in the publication
0.059
benzene-1,3-disulfonate
-
pH and temperature not specified in the publication
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
2 - 10
-
pH 2.0: about 40% of maximal activity of soluble enzyme, about 60% of maximal activity of immobilized enzyme, pH 10.0: about 80% of maximal activity of immobilized enzyme, about 40% of maximal activity of soluble enzyme
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20 - 70
20 - 70
-
20°C: about 65% of maximal activity, 70°C: about 30% of maximal activity, soluble enzyme
20 - 70
-
more than 80% of maximal activity is observed between 20°C and 70°C, immobilized enzyme
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
physiological function
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)
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
physiological function
-
glutamate racemase catalyzes stereoinversion at the Calpha of glutamate and is a source of D-glutamate in bacteria, an essential component of the peptidoglycan layer of the bacterial cell walls
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
monomer
determined by gel filtration, BsGR is a monomeric enzyme, which dimerizes in the presence of either 10 mM D- or L-glutamate
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
T76A
production by site-directed mutagenesis, strong RacE-glutamate carbanion interaction energy is notably dissipated with the mutant
additional information
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
additional information
-
disruption of enzyme gene yrpC, no effect on growth or production of poly-gamma-glutamte. Disruption of enzyme gene glr, cells are only viable when an exogenous gene copy is present on a plasmid. Glr gene product is responsible for supply of D-glutamate both to the synthesis of peptidoglycan and poly-gamma-glutamate
additional information
-
enzyme knockout mutants, gene racE is essential for growth in rich medium but dispensable for growth in minimal medium. YrpC gene is expressed only in minimal medium. Neither gene is required for synthesis of poly-gamma-DL-glutamate. RacE or yrpC mutant cells accumulate signifcant amounts of D- but not L-glutamate. RacE/yrpC double mutant shows severely impaired D-amino acid utilization
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
50
pH 8.0, 0.1 M Tris-HCl buffer containing 10% glycerol, retains 90% of its activity after 10 min
60
-
pH 7, 60 min, about 25% loss of activity of soluble and immobilized enzyme
80
-
pH 7, 60 min, about 20% loss of activity of immobilized enzyme, about 70% loss of activity of soluble enzyme
90
-
pH 7, 60 min, 50% loss of activity of immobilized enzyme, about 75% loss of activity of soluble enzyme
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
stabilized by immobilization to Chitopearl 2505 or Chitopearl 2605. The enzyme remains active at 4°C for 30 days, remaining activity is 32%
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene murI, recombinant His-tagged enzyme in Escherichia coli strain BL21(DE3)
overexpression in BL21(DE3) Escherichia coli as fusion protein bearing an N-terminal hexahistidine tag
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
recombinant wild type and mutant proteins are expressed in Escherichia coli cells
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
drug development
enzyme is an attractive target for the development of antibacterial agents
drug development
glutamate racemase is an attractive target for the design of antibacterial agents
synthesis
-
production of D-glutamate from L-glutamate with glutamate racemase and L-glutamate oxidase from Streptomyces sp. X119-6. Both enzymes are highly stabilized by immobilization
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Ashiuchi, M.; Tani, K.; Soda, K.; Misono, H.
Properties of glutamate racemase from Bacillus subtilis IFO 3336 producing poly-gamma-glutamate
J. Biochem.
123
1156-1163
1998
Bacillus subtilis, Bacillus subtilis (P94556), Bacillus subtilis CU741, Pediococcus pentosaceus
Oikawa, T.; Watanabe, M.; Makiura, H.; Kusakabe, H.; Yamade, K.; Soda, K.
Production of D-glutamate from L-glutamate with glutamate racemase and L-glutamate oxidase
Biosci. Biotechnol. Biochem.
63
2168-2173
1999
Bacillus subtilis
Taal, M.A.; Sedelnikova, S.E.; Ruzheinikov, S.N.; Baker, P.J.; Rice, D.W.
Expression, purification and preliminary X-ray analysis of crystals of Bacillus subtilis glutamate racemase
Acta Crystallogr. Sect. D
60
2031-2034
2004
Bacillus subtilis
Kada, S.; Nanamiya, H.; Kawamura, F.; Horinouchi, S.
Glr, a glutamate racemase, supplies D-glutamate to both peptidoglycan synthesis and poly-gamma-glutamate production in gamma-PGA-producing Bacillus subtilis
FEMS Microbiol. Lett.
236
13-20
2004
Bacillus subtilis
Kimura, K.; Tran, L.S.; Itoh, Y.
Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis
Microbiology
150
2911-2920
2004
Bacillus subtilis
Ruzheinikov, S.N.; Taal, M.A.; Sedelnikova, S.E.; Baker, P.J.; Rice, D.W.
Substrate-induced conformational changes in Bacillus subtilis glutamate racemase and their implications for drug discovery
Structure
13
1707-1713
2005
Bacillus subtilis
Potrykus, J.; Flemming, J.; Bearne, S.
Kinetic characterization and quaternary structure of glutamate racemase from the periodontal anaerobe Fusobacterium nucleatum
Arch. Biochem. Biophys.
491
16-24
2009
Bacillus subtilis (P94556), Bacillus subtilis, Fusobacterium nucleatum subsp. nucleatum (Q8REE6)
Puig, E.; Mixcoha, E.; Garcia-Viloca, M.; Gonzlez-Lafont, A.; Lluch, J.
How the substrate D-glutamate drives the catalytic action of bacillus subtilis glutamate racemase
J. Am. Chem. Soc.
131
3509-3521
2009
Bacillus subtilis
Spies, M.; Reese, J.; Dodd, D.; Pankow, K.; Blanke, S.; Baudry, J.
Determinants of catalytic power and ligand binding in glutamate racemase
J. Am. Chem. Soc.
131
5274-5284
2009
Bacillus subtilis (Q6L876), Bacillus subtilis
Whalen, K.L.; Pankow, K.L.; Blanke, S.R.; Spies, M.A.
Exploiting enzyme plasticity in virtual screening: high efficiency inhibitors of glutamate racemase
ACS Med. Chem. Lett.
1
9-13
2010
Bacillus subtilis
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
ACS Cent. Sci.
1
364-373
2015
Bacillus subtilis (P94556), Bacillus subtilis 168 (P94556)
Prosser, G.A.; Rodenburg, A.; Khoury, H.; de Chiara, C.; Howell, S.; Snijders, A.P.; de Carvalho, L.P.
Glutamate racemase is the primary target of beta-chloro-D-alanine in Mycobacterium tuberculosis
Antimicrob. Agents Chemother.
60
6091-6099
2016
Bacillus subtilis (P94556), Bacillus subtilis 168 (P94556), Mycobacterium tuberculosis (P9WPW9), Mycobacterium tuberculosis, Mycobacterium tuberculosis ATCC 25618 / H37Rv (P9WPW9)
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