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Literature summary for 5.1.1.3 extracted from

  • Poen, S.; Nakatani, Y.; Opel-Reading, H.K.; Lasse, M.; Dobson, R.C.; Krause, K.L.
    Exploring the structure of glutamate racemase from Mycobacterium tuberculosis as a template for anti-mycobacterial drug discovery (2016), Biochem. J., 473, 1267-1280 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene murI, recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Mycolicibacterium smegmatis
gene murI, recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) pLysS Mycobacterium tuberculosis

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant enzyme complexed with D-glutamate, hanging drop vapour diffusion method, mixing of 0.002 ml of 8 mg/ml protein in 20 mM Tris HCl, pH 8.0, and 1 mM D-glutamate, with 0.002 ml of 18% PEG3350, 0.2 M NaI, 4°C, 4 days, X-ray diffraction structure determination and analysis at 1.76 A resolution Mycolicibacterium smegmatis
purified recombinant enzyme complexed with D-glutamate, microbatch-underoil method, mixing of 0.002 ml of 5 mg/ml protein in 20 mM Tris HCl, pH 8.0, and 1 mM D-glutamate, with 0.001 ml of crystallizing solution containing 0.1 M HEPES, pH 7.5, and 20% PEG 10000, 18°C, 14 days, X-ray diffraction structure determination and analysis at 2.30 A resolution Mycobacterium tuberculosis

Protein Variants

Protein Variants Comment Organism
D26R/R105A/G194E site-directed mutagenesis, mutation of the interface affording a soluble and active enzyme Mycobacterium tuberculosis
D26R/R105A/G194R site-directed mutagenesis, mutation of the interface affording a soluble and active enzyme Mycobacterium tuberculosis
additional information site-directed mutagenesis of the enzyme's interface affording a soluble and active enzyme Mycolicibacterium smegmatis

Inhibitors

Inhibitors Comment Organism Structure
additional information inhibitor screening, compounds designed to target other glutamate racemases have been screened but do not inhibit mycobacterial MurI, suggesting that a different drug design effort will be needed to develop inhibitors. A distinct type of MurI dimer arrangement is observed in both structures, and this arrangement becomes the third biological dimer geometry for MurI found Mycobacterium tuberculosis
additional information inhibitor screening, compounds designed to target other glutamate racemases have been screened but do not inhibit mycobacterial MurI, suggesting that a different drug design effort will be needed to develop inhibitors. A distinct type of MurI dimer arrangement is observed in both structures, and this arrangement becomes the third biological dimer geometry for MurI found Mycolicibacterium smegmatis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
54200
-
recombinant enzyme, gel filtration Mycobacterium tuberculosis
59500
-
recombinant enzyme, gel filtration Mycolicibacterium smegmatis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-glutamate Mycobacterium tuberculosis
-
D-glutamate
-
r
L-glutamate Mycolicibacterium smegmatis
-
D-glutamate
-
r
L-glutamate Mycobacterium tuberculosis ATCC 25618 / H37Rv
-
D-glutamate
-
r
L-glutamate Mycolicibacterium smegmatis ATCC 700084 / mc(2)155
-
D-glutamate
-
r

Organism

Organism UniProt Comment Textmining
Mycobacterium tuberculosis P9WPW9
-
-
Mycobacterium tuberculosis ATCC 25618 / H37Rv P9WPW9
-
-
Mycolicibacterium smegmatis A0R1X0
-
-
Mycolicibacterium smegmatis ATCC 700084 / mc(2)155 A0R1X0
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag removal by TEV protease, and gel filtration, to over 95% purity Mycobacterium tuberculosis
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag removal by TEV protease, and gel filtration, to over 95% purity Mycolicibacterium smegmatis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
D-glutamate D-Glu binding structure, overview Mycobacterium tuberculosis L-glutamate
-
r
D-glutamate D-Glu binding structure, overview Mycolicibacterium smegmatis L-glutamate
-
r
D-glutamate D-Glu binding structure, overview Mycobacterium tuberculosis ATCC 25618 / H37Rv L-glutamate
-
r
D-glutamate D-Glu binding structure, overview Mycolicibacterium smegmatis ATCC 700084 / mc(2)155 L-glutamate
-
r
L-glutamate
-
Mycobacterium tuberculosis D-glutamate
-
r
L-glutamate
-
Mycolicibacterium smegmatis D-glutamate
-
r
L-glutamate
-
Mycobacterium tuberculosis ATCC 25618 / H37Rv D-glutamate
-
r
L-glutamate
-
Mycolicibacterium smegmatis ATCC 700084 / mc(2)155 D-glutamate
-
r

Subunits

Subunits Comment Organism
homodimer 2 * 28900, about, sequence calcuation and analytical ultracentrifugation Mycobacterium tuberculosis
homodimer 2 * 29500, about, sequence calcuation and analytical ultracentrifugation Mycolicibacterium smegmatis
More the active site is located in a surface-exposed cleft that is formed at the interface of domains 1 and 2, subunit interaction analysis Mycobacterium tuberculosis
More the active site is located in a surface-exposed cleft that is formed at the interface of domains 1 and 2, subunit interaction analysis Mycolicibacterium smegmatis

Synonyms

Synonyms Comment Organism
MurI
-
Mycobacterium tuberculosis
MurI
-
Mycolicibacterium smegmatis

General Information

General Information Comment Organism
additional information the mycobacterial MurI dimer is tightly associated, with a KD in the nanomolar range. The enzyme binds D- and L-glutamate specifically, but is inactive in solution unless the dimer interface is mutated. Enzyme structure analysis, active site structure and substrate binding structure, overview Mycobacterium tuberculosis
additional information the mycobacterial MurI dimer is tightly associated, with a KD in the nanomolar range. The enzyme binds D- and L-glutamate specifically, but is inactive in solution unless the dimer interface is mutated. Enzyme structure analysis, active site structure and substrate binding structure, overview Mycolicibacterium smegmatis
physiological function glutamate racemase (MurI) is responsible for providing D-glutamate for peptidoglycan biosynthesis in bacteria Mycobacterium tuberculosis
physiological function glutamate racemase (MurI) is responsible for providing D-glutamate for peptidoglycan biosynthesis in bacteria Mycolicibacterium smegmatis