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Literature summary for 5.1.1.3 extracted from

  • 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 (2016), Antimicrob. Agents Chemother., 60, 6091-6099 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 5.1.1.3

Cloned(Commentary)

Cloned (Comment) Organism
gene murI, recombinant His-tagged enzyme in Escherichia coli strain BL21(DE3) Bacillus subtilis
gene murI, recombinant His-tagged enzyme in Escherichia coli strain BL21(DE3) Mycobacterium tuberculosis

Inhibitors

Inhibitors Comment Organism Structure
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 Bacillus subtilis
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 modifies MtMurI in vitro and in vivo Mycobacterium tuberculosis
beta-chloro-L-alanine BCLA, modifies BsMurI predominantly at C74, the cysteine residue responsible for deprotonation of an incoming D-Glu substrate Bacillus subtilis
additional information no inhibition by beta-fluoroalanine (BFA, racemic mixture) and O-acetyl-D-serine (OADS) Bacillus subtilis
additional information no inhibition by beta-fluoroalanine (BFA, racemic mixture) and O-acetyl-D-serine (OADS). MtMurI adopts a unique oligomeric configuration and contains distinct active-site architectural differences from other MurI orthologues and that dimer interface mutations are required to introduce catalytic capability. Conventional MurI inhibitors are inactive against this recombinant form of mycobacterial MurI, the selective inhibitory activity of BCDA against MtMurI may arise from unique protein-ligand interactions unseen in orthologous MurIs. MsMurI results demonstrate these same structural features and yet BCDA does not appear to act via MurI in this bacterium Mycobacterium tuberculosis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5
-
 D-glutamate pH 7.6, 37°C Mycobacterium tuberculosis
37
-
 L-glutamate pH 7.6, 37°C Mycobacterium tuberculosis

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 Mycobacterium tuberculosis
-
 D-glutamate
-
 r
L-glutamate Bacillus subtilis 168
-
 D-glutamate
-
 r
L-glutamate Mycobacterium tuberculosis ATCC 25618 / H37Rv
-
 D-glutamate
-
 r

Organism

Organism UniProt Comment Textmining
Bacillus subtilis P94556
-
-
Bacillus subtilis 168 P94556
-
-
Mycobacterium tuberculosis P9WPW9
-
-
Mycobacterium tuberculosis ATCC 25618 / H37Rv P9WPW9
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage Bacillus subtilis
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage Mycobacterium tuberculosis

Reaction

Reaction Comment Organism Reaction ID
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 Mycobacterium tuberculosis
a
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) Bacillus subtilis
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
D-glutamate
-
 Mycobacterium tuberculosis L-glutamate
-
 r
D-glutamate
-
 Mycobacterium tuberculosis ATCC 25618 / H37Rv L-glutamate
-
 r
L-glutamate
-
 Bacillus subtilis D-glutamate
-
 r
L-glutamate
-
 Mycobacterium tuberculosis D-glutamate
-
 r
L-glutamate
-
 Bacillus subtilis 168 D-glutamate
-
 r
L-glutamate
-
 Mycobacterium tuberculosis ATCC 25618 / H37Rv D-glutamate
-
 r

Synonyms

Synonyms Comment Organism
MurI
-
 Bacillus subtilis
MurI
-
 Mycobacterium tuberculosis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Bacillus subtilis
37
-
 assay at Mycobacterium tuberculosis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
12.6
-
 D-glutamate pH 7.6, 37°C Mycobacterium tuberculosis
226.4
-
 L-glutamate pH 7.6, 37°C Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.6
-
 assay at Bacillus subtilis
7.6
-
 assay at Mycobacterium tuberculosis

Cofactor

Cofactor Comment Organism Structure
additional information glutamate racemase (MurI) is a pyridoxal 5'-phosphate-independent racemase Bacillus subtilis
additional information glutamate racemase (MurI) is a pyridoxal 5'-phosphate-independent racemase Mycobacterium tuberculosis

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.13
-
 beta-Chloro-D-alanine pH 7.6, 37°C Mycobacterium tuberculosis
0.23
-
 beta-Chloro-D-alanine pH 7.6, 37°C Bacillus subtilis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
6.12
-
 L-glutamate pH 7.6, 37°C Mycobacterium tuberculosis
8.4
-
 D-glutamate pH 7.6, 37°C Mycobacterium tuberculosis