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Literature summary 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

Cloned(Commentary)

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

Inhibitors

EC Number Inhibitors Comment Organism Structure
5.1.1.1 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. Glutamate racemase (MurI) is a pyridoxal 5'-phosphate-independent racemase and is therefore unable to undergo the same mechanism of inhibition as Alr with BCDA Bacillus subtilis
5.1.1.1 beta-Chloro-D-alanine BCDA, the compund is a very poor inhibitor of recombinant Mycobacterium tuberculosis Alr, despite having potent antituberculosis activity, 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. Glutamate racemase (MurI) is a pyridoxal 5'-phosphate-independent racemase and is therefore unable to undergo the same mechanism of inhibition as Alr with BCDA Mycobacterium tuberculosis
5.1.1.3 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
5.1.1.3 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
5.1.1.3 beta-chloro-L-alanine BCLA, modifies BsMurI predominantly at C74, the cysteine residue responsible for deprotonation of an incoming D-Glu substrate Bacillus subtilis
5.1.1.3 additional information no inhibition by beta-fluoroalanine (BFA, racemic mixture) and O-acetyl-D-serine (OADS) Bacillus subtilis
5.1.1.3 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
5.1.1.1 3.7
-
L-alanine pH 7.6, 37°C Mycobacterium tuberculosis
5.1.1.1 5.9
-
L-alanine pH 7.6, 37°C Bacillus subtilis
5.1.1.3 1.5
-
D-glutamate pH 7.6, 37°C Mycobacterium tuberculosis
5.1.1.3 37
-
L-glutamate pH 7.6, 37°C Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
5.1.1.1 L-alanine Bacillus subtilis
-
D-alanine
-
r
5.1.1.1 L-alanine Mycobacterium tuberculosis
-
D-alanine
-
r
5.1.1.1 L-alanine Bacillus subtilis 168
-
D-alanine
-
r
5.1.1.1 L-alanine Mycobacterium tuberculosis ATCC 25618 / H37Rv
-
D-alanine
-
r
5.1.1.3 L-glutamate Bacillus subtilis
-
D-glutamate
-
r
5.1.1.3 L-glutamate Mycobacterium tuberculosis
-
D-glutamate
-
r
5.1.1.3 L-glutamate Bacillus subtilis 168
-
D-glutamate
-
r
5.1.1.3 L-glutamate Mycobacterium tuberculosis ATCC 25618 / H37Rv
-
D-glutamate
-
r

Organism

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

Purification (Commentary)

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

Reaction

EC Number Reaction Comment Organism Reaction ID
5.1.1.3 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
5.1.1.3 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

Substrates and Products (Substrate)

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

Synonyms

EC Number Synonyms Comment Organism
5.1.1.1 MurI
-
Bacillus subtilis
5.1.1.1 MurI
-
Mycobacterium tuberculosis
5.1.1.3 MurI
-
Bacillus subtilis
5.1.1.3 MurI
-
Mycobacterium tuberculosis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
5.1.1.1 37
-
assay at Bacillus subtilis
5.1.1.1 37
-
assay at Mycobacterium tuberculosis
5.1.1.3 37
-
assay at Bacillus subtilis
5.1.1.3 37
-
assay at Mycobacterium tuberculosis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.1.1.1 37
-
L-alanine pH 7.6, 37°C Mycobacterium tuberculosis
5.1.1.1 1190
-
L-alanine pH 7.6, 37°C Bacillus subtilis
5.1.1.3 12.6
-
D-glutamate pH 7.6, 37°C Mycobacterium tuberculosis
5.1.1.3 226.4
-
L-glutamate pH 7.6, 37°C Mycobacterium tuberculosis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
5.1.1.1 7.6
-
assay at Bacillus subtilis
5.1.1.1 7.6
-
assay at Mycobacterium tuberculosis
5.1.1.3 7.6
-
assay at Bacillus subtilis
5.1.1.3 7.6
-
assay at Mycobacterium tuberculosis

Cofactor

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

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
5.1.1.1 0.13
-
beta-Chloro-D-alanine pH 7.6, 37°C Mycobacterium tuberculosis
5.1.1.1 0.23
-
beta-Chloro-D-alanine pH 7.6, 37°C Bacillus subtilis
5.1.1.3 0.13
-
beta-Chloro-D-alanine pH 7.6, 37°C Mycobacterium tuberculosis
5.1.1.3 0.23
-
beta-Chloro-D-alanine pH 7.6, 37°C Bacillus subtilis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
5.1.1.1 10
-
L-alanine pH 7.6, 37°C Mycobacterium tuberculosis
5.1.1.1 201.7
-
L-alanine pH 7.6, 37°C Bacillus subtilis
5.1.1.3 6.12
-
L-glutamate pH 7.6, 37°C Mycobacterium tuberculosis
5.1.1.3 8.4
-
D-glutamate pH 7.6, 37°C Mycobacterium tuberculosis