Information on EC 3.5.1.103 - N-acetyl-1-D-myo-inositol-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase

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The expected taxonomic range for this enzyme is: Mycobacterium

EC NUMBER
COMMENTARY hide
3.5.1.103
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RECOMMENDED NAME
GeneOntology No.
N-acetyl-1-D-myo-inositol-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol + H2O = 1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol + acetate
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
mycothiol biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
1-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol acetylhydrolase
This enzyme is considered the key enzyme and rate limiting step in the mycothiol biosynthesis pathway [1]. In addition to acetylase activity, the enzyme possesses weak activity of EC 3.5.1.115, mycothiol S-conjugate amidase, and shares sequence similarity with that enzyme [2]. The enzyme requires a divalent transition metal ion for activity, believed to be Zn2+ [3].
CAS REGISTRY NUMBER
COMMENTARY hide
340703-87-9
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol + H2O
1-(2-amino-2-deoxy-alpha-D-glucopyranoside)-1D-myo-inositol + acetate
show the reaction diagram
1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside + H2O
1-(2-amino-2-deoxy-alpha-D-glucopyranoside)-1D-myo-inositol + acetate
show the reaction diagram
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key enzyme in mycothiol biosynthesis. Mycothiol is the major low molecular weight thiol in actinomycetes and is essential for growth of Mycobacterium tuberculosis
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-
?
1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol + H2O
1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol + acetate
show the reaction diagram
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-
-
-
?
cyclohexyl-2-acetamido-2-deoxy-1-thio-alpha-D-glucopyranoside + H2O
cyclohexyl-2-amino-2-deoxy-1-thio-alpha-D-glucopyranoside + acetate
show the reaction diagram
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6% of the activity with 1-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol
-
-
?
cyclohexyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside + H2O
cyclohexyl-2-amino-2-deoxy-alpha-D-glucopyranoside + acetate
show the reaction diagram
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6% of the activity with 1-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol
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-
?
N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside + H2O
1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside + acetate
show the reaction diagram
-
-
-
-
?
N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-D-glucopyranoside + H2O
1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol + acetate
show the reaction diagram
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molecular docking analysis of the MshB active site binding pocket, overview
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-
?
N-acetyl-1D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside + H2O
1D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside + acetate
show the reaction diagram
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-
-
-
?
N-acetyl-D-glucosamine + H2O
acetate + D-glucosamine
show the reaction diagram
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commercially available alternate minimal substrate, MshB is 100fold less active towards this compound than toward the natural substrate
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-
?
N-acetyl-D-glucosamine + H2O
D-glucosamine + acetate
show the reaction diagram
N-deacetyl-N-formylmycothiol-monobromobimane conjugate + H2O
?
show the reaction diagram
-
-
-
-
?
phenyl 2-acetamido-2-deoxy-1-thio-alpha-D-glucopyranoside + H2O
phenyl 2-amino-2-deoxy-1-thio-alpha-D-glucopyranoside + acetate
show the reaction diagram
additional information
?
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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-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol + H2O
1-(2-amino-2-deoxy-alpha-D-glucopyranoside)-1D-myo-inositol + acetate
show the reaction diagram
1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside + H2O
1-(2-amino-2-deoxy-alpha-D-glucopyranoside)-1D-myo-inositol + acetate
show the reaction diagram
-
key enzyme in mycothiol biosynthesis. Mycothiol is the major low molecular weight thiol in actinomycetes and is essential for growth of Mycobacterium tuberculosis
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-
?
1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol + H2O
1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol + acetate
show the reaction diagram
-
-
-
-
?
N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside + H2O
1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside + acetate
show the reaction diagram
-
-
-
-
?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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enzyme has a strong preference for the Fe2+ cofactor under anaerobic conditions, regardless of the metal ion content of the medium. The preferred cofactor changes between Zn2+ and Fe2+ depending on the metal ion content of the medium when MshB is purified under aerobic conditions
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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0.1 mM, 82% inhibition of the enzyme isolated on the Ni-affinity column. 0.1mM 1,10-phenanthroline produces no signifiant inhibition of the enzyme isolated on the Zn-affinity column and 10% activity remains after treatment with 1 mM 1,10-phenanthroline. MshB activity lost by incubation of the Ni enzyme with 1,10-phenanthroline can be restored following removal of 1,10-phenanthroline by incubation with 0.1 mM Zn2+, Ni2+, Mn2+, or Co2+, the latter promoting the highest activity, but Ca2+ and Mg2+ produce no restoration of activity; inhibition of the Zn enzyme by 1,10-phenanthroline is slower or less complete than inhibition of the Ni enzyme. MshB activity lost by incubation of the Ni enzyme with 1,10-phenanthroline can be restored following removal of 1,10-phenanthroline by incubation with 0.1 mM Zn2+, Ni2+, Mn2+, or Co2+, the latter promoting the highest activity. Ca2+ and Mg2+ produce no restoration of activity
benzyl 2-deoxy-2-acetamido-1-thio-beta-D-glucopyranoside
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0.5 mM, 13% inhibition
cyclohexyl (2''R),(2''S)-3,3''-anhydro-2-deoxy-2-C-(2'',3''-dihydroxypropyl)-alpha-D-glucopyranoside
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0.5 mM, 6.6% inhibition
cyclohexyl 2-deoxy-2-C-(2'',3''-epoxypropyl)-alpha-D-glucopyranoside
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0.5 mM, 19.7% inhibition
cyclohexyl 2-deoxy-2-C-(2''-hydroxypropyl)-alpha-D-glucopyranoside
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0.5 mM, 11.4% inhibition
cyclohexyl 2-deoxy-2-C-(2''-oxopropyl)-a-D-glucopyranoside
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0.5 mM, 6.7% inhibition
cyclohexyl-2-chloroacetamido-2-deoxy-1-thio-alpha-D-glucopyranoside
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0.5 mM, 15% inhibition
NaF
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uncompetitive inhibitor, consistent with a metal-water/hydroxide functioning as the reactive nucleophile in the catalytic mechanism
phenyl-2-acetamido-2-deoxy-1-thio-alpha-D-glucopyranoside
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0.5 mM, 6% inhibition
phenyl-2-deoxy-2-acetamido-1-thio-beta-D-glucopyranoside
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0.5 mM, 15% inhibition
phenyl-2-deoxy-2-[3'-(8''-hydroxy-3''-methyl-1'',4''-dioxo-1'',4''-dihydronaphthalen-2''-yl)butanamido]-1-thio-alpha-D-glucopyranoside
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0.5 mM, 81.6% inhibition
phenyl-2-deoxy-2-[3'-(8''-hydroxy-3''-methyl-1'',4''-dioxo-1'',4''-dihydronaphthalen-2''-yl)pentanamido]-1-thio-alpha-D-glucopyranoside
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0.5 mM, 81.4% inhibition
phenyl-2-deoxy-2-[3'-(8''-hydroxy-3''-methyl-1'',4''-dioxo-1'',4''-dihydronaphthalen-2''-yl)propanamido]-1-thio-alpha-D-glucopyranoside
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0.5 mM, 57.4% inhibition
additional information
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no inhibition is observed with 0.1 M 1,7-phenanthroline
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,7-phenanthroline
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1 mM, slight enhancement of activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.34 - 0.348
1-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol
9 - 400
N-acetyl-D-glucosamine
0.34
N-deacetyl-N-formylmycothiol-monobromobimane conjugate
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pH 7.4, 37C
1.695
phenyl-2-acetamido-2-deoxy-1-thio-alpha-D-glucopyranoside
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.24 - 0.49
1-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol
0.0048 - 2.1
N-acetyl-D-glucosamine
0.49
N-deacetyl-N-formylmycothiol-monobromobimane conjugate
Mycobacterium tuberculosis
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pH 7.4, 37C
0.12
phenyl-2-acetamido-2-deoxy-1-thio-alpha-D-glucopyranoside
Mycobacterium tuberculosis
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pH 7.5, 37C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.68 - 1.44
1-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-1D-myo-inositol
19037
0.00009 - 0.2
N-acetyl-D-glucosamine
300
0.07
phenyl-2-acetamido-2-deoxy-1-thio-alpha-D-glucopyranoside
Mycobacterium tuberculosis
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pH 7.5, 37C
18988
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32000
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2 * 32000, SDS-PAGE; 2 * 32000, the enzyme appears to behave as a dimer at normal ionic strength but may associate to a tetramer at low ionic strength, SDS-PAGE; 4 * 32000, the enzyme appears to behave as a dimer at normal ionic strength but may associate to a tetramer at low ionic strength, SDS-PAGE
33423
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2 * 33423, the enzyme appears to behave as a dimer at normal ionic strength but may associate to a tetramer at low ionic strength, calculated from sequence; 4 * 33423, the enzyme appears to behave as a dimer at normal ionic strength but may associate to a tetramer at low ionic strength, calculated from sequence
79000
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dimer, gel filtration; dimer, the enzyme behaves as a dimer at normal ionic strength but may associate to a tetramer at low ionic strength, gel filtration
158000
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tetramer, gel filtration; tetramer, the enzyme behaves as a dimer at normal ionic strength but may associate to a tetramer at low ionic strength, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals prepared in the presence of beta-octylglucoside as a key additive, are suitable for high-resolution X-ray structural analysis. The crystals are orthorhombic, with unit-cell parameters a = 71.69, b = 83.74, c = 95.65 A, space group P2(1)2(1)2(1) and two molecules in the asymmetric unit. Collection of X-ray diffraction data to 1.9 A resolution
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hanging-drop vapour-diffusion method, crystallized both in native and SeMet-substituted forms; structure, determined at 1.9 A resolution by X-ray crystallography; the structure, determined at 1.9 A resolution by X-ray crystallography, reveals an alpha/beta fold in which helices pack against a seven-stranded mostly parallel beta-sheet
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purified recombinant enzyme, from 0.1 M sodium acetate, 0.2 M ammonium sulfate, 25% PEG 4000 pH 4.6, with glycerol and the reaction product acetate in the active site, X-ray diffraction structure determination and analysis at 1.94 A resolution, modelling
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vapor-diffusion method; vapor-diffusion method with a mother liquor consisting of 15% polyethylene glycol 4000, 50 mm Tris-HCl (pH 8.0), 0.1 m Mg(NO3)2, 6% 1,6-hexanediol, and 10% ethylene glycol. A 1:2 ratio of protein solution (6 mg/ml) to mother liquor is mixed and left for vapor equilibration. Triclinic crystals form after approximately 1 week at room temperature
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression in Escherichia coli both in native and SeMet-substituted forms
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expression in Escherichia coli; Rv1170 is cloned to contain a C-terminal His-6 tag to facilitate purifiation, expression in Escherichia coli
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recombinant enzyme expression
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D146A
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below 0.4% of wild-type activity
D15A
-
0.5% of wild-type activity
H144A
-
1.0% of wild-type activity
Y142A
-
6.8% of wild-type activity
Y142F
-
5.3% of wild-type activity
Y142Q
-
5.9% of wild-type activity
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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fluorescence-based assay for measuring N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase activity that does not require HPLC analysis and can be carried out in multiwell plates. The fluorescamine-based assay can be used to determine the steady-state parameters for the deacetylation of N-acetyl-glucosamine by MshB
drug development
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enzyme MshB is a target for the discovery of drugs to treat tuberculosis
medicine
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