Information on EC 6.3.1.13 - L-cysteine:1D-myo-inositol 2-amino-2-deoxy-alpha-D-glucopyranoside ligase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
6.3.1.13
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RECOMMENDED NAME
GeneOntology No.
L-cysteine:1D-myo-inositol 2-amino-2-deoxy-alpha-D-glucopyranoside ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol + L-cysteine + ATP = 1-O-[2-(L-cysteinamido)-2-deoxy-alpha-D-glucopyranosyl]-1D-myo-inositol + AMP + diphosphate
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
mycothiol biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
L-cysteine:1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol ligase (AMP-forming)
This enzyme is a key enzyme in the biosynthesis of mycothiol, a small molecular weight thiol found in Mycobacteria spp. and other actinomycetes. Mycothiol plays a fundamental role in these organisms by helping to provide protection from the effects of reactive oxygen species and electrophiles, including many antibiotics. The enzyme may represent a novel target for new classes of antituberculars [2].
CAS REGISTRY NUMBER
COMMENTARY hide
442171-76-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene mshC
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Manually annotated by BRENDA team
strain mc2 155, gene mshC, formerly gene cys2, 2 different splicing variants due to a second start codon
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Manually annotated by BRENDA team
strain Rv2130c, gene mshC
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Manually annotated by BRENDA team
RHA1, generation of an MshC mutant (RHA1_022) in RHA1
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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mutants defective in mycothiol biosynthesis show mutations in genes coding for the glycosyltransferase (mshA) or the cysteine ligase (mshC). These mutants show low-level resistance to isoniazid but are highly resistant to ethionamide, mutations in mycothiol biosynthesis genes may contribute to isoniazid or ethionamide resistance across mycobacterial species
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol + L-cysteine + ATP
1-O-[2-(L-cysteinamido)-2-deoxy-alpha-D-glucopyranosyl]-1D-myo-inositol + AMP + diphosphate
show the reaction diagram
additional information
?
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penultimate enzyme in the mycothiol biosynthetic pathway
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol + L-cysteine + ATP
1-O-[2-(L-cysteinamido)-2-deoxy-alpha-D-glucopyranosyl]-1D-myo-inositol + AMP + diphosphate
show the reaction diagram
additional information
?
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penultimate enzyme in the mycothiol biosynthetic pathway
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5'-O-[N-(L-cysteinyl)sulfamonyl]adenosine
cysteinyl sulfamoyl adenosine
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selective, discrimination of aminoacyl-t-RNA synthetase inhibitors occurs at the amino acid moiety
dequalinium chloride
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ATP-competitive inhibitor of MshC, binds MshC with a KD of 0.22 microM, and inhibits the growth of Mycobacterium tuberculosis under aerobic and anaerobic conditions with minimum inhibitory and anaerobic bactericidal concentrations of 1.2 and 0.3 microg/ml, respectively
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.072
1D-myo-inositol 2-amino-2-deoxy-alpha-D-glucopyranoside
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pH 7.5, 37°C
0.16
1D-myo-inositol 2-amino-2-deoxy-alpha-D-glycopyranoside
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pH 7.8, 25°C, recombinant enzyme
1.8
ATP
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pH 7.8, 25°C, recombinant enzyme
0.04 - 0.1
L-cysteine
additional information
additional information
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steady-state and pre-steady-state kinetic analysis, single-turnover reactions of the first and second half reactions
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.15
1D-myo-inositol 2-amino-2-deoxy-alpha-D-glycopyranoside
Mycobacterium smegmatis
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pH 7.8, 25°C, recombinant enzyme
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00031
5'-O-[N-(L-cysteinyl)sulfamonyl]adenosine
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versus ATP
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00005
cysteinyl sulfamoyl adenosine
Mycobacterium tuberculosis
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pH not specified in the publication, temperature not specified in the publication
0.024
dequalinium
Mycobacterium tuberculosis
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pH 8.0, 22°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.009
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purified recombinant GST-tagged MshC
0.025
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purified recombinant maltose binding protein-MshC
0.054
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purified enzyme
0.15
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purified recombinant enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40700
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recombinant His-tagged enzyme, gel filtration
47000
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1 * 47000, recombinant His-tagged enzyme, 1 * 47562, amino acid sequence calculation
47562
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1 * 47000, recombinant His-tagged enzyme, 1 * 47562, amino acid sequence calculation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 47000, recombinant His-tagged enzyme, 1 * 47562, amino acid sequence calculation
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
incubation of the enzyme with the cysteinyl adenylate analogue, 5'-O-[N-(L-cysteinyl)-sulfamonyl]adenosine, followed by a 24 h limited trypsin proteolysis yields an enzyme preparation that readily crystallizes, 1.6 A crystal structure
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2400fold, 2 peaks
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recombinant enzyme from mshC-deficient mutant strain I64 of Mycobacterium smegmatis by ammonium sulfate fractionation, anion exchange chromatography, hydroxyapatite chromatography, and gel filtration
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recombinant GST-tagged MshC and recombinant maltose binding protein-MshC by affinity chromatography on glutathione and amylose resins, respectively
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recombinant His-tagged enzyme from Escherichia coli strain BL21 by nickel affinity chromatography and gel filtration to about 95% purity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, expression in Escherichia coli BL21(DE3) as His-tagged enzyme
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DNA and amino acid sequence determination and analysis, expression in Escherichia coli BL21(DE3) as N-terminally His-tagged enzyme
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expression of His-tagged enzyme in Escherichia coli strain BL21
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gene mshC, construction of three N-terminal-MshC fusion proteins where N-terminal tags include the B1 domain of 1. streptococcal protein G to give GB1-MshC, 2. glutathione-S-transferase to give GST-MshC, and 3. maltose binding protein to give MBP-MshC, for expression in M. smegmatis, expression in enzyme-deficient mutant strain mc2155, i.e. I64 L205P, optimization of recombinant enzyme expression, overview
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gene mshC, expression in the mshC-deficient mutant strain I64 of Mycobacterium smegmatis, expression as insoluble protein in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
del D86
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naturally occuring mutation, del bp 255-257, the mutant shows 6.9% activity compared to the wild-type
G54S
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naturally occuring mutation, inactive mutant
P47R
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naturally occuring mutation, inactive mutant
additional information
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mutant mc24980attB::pMV361::mshCBo shows 169% activity compared to the wild-type enzyme
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
pharmacology
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