Information on EC 2.3.1.4 - glucosamine-phosphate N-acetyltransferase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.3.1.4
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RECOMMENDED NAME
GeneOntology No.
glucosamine-phosphate N-acetyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
acetyl-CoA + D-glucosamine 6-phosphate = CoA + N-acetyl-D-glucosamine 6-phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acyl group transfer
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Amino sugar and nucleotide sugar metabolism
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UDP-N-acetyl-D-galactosamine biosynthesis II
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UDP-N-acetyl-D-glucosamine biosynthesis II
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SYSTEMATIC NAME
IUBMB Comments
acetyl-CoA:D-glucosamine-6-phosphate N-acetyltransferase
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CAS REGISTRY NUMBER
COMMENTARY hide
9031-91-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
bi-functional glucosamine-1-phosphate acetyltransferase/N-acetylglucosamine-1-phosphate uridyltransferase
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-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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Uniprot
Manually annotated by BRENDA team
strain 5297a
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-
Manually annotated by BRENDA team
mutant NB208 exhibiting a temperature-sensitive defect in root elongation, NB208 is a GNA1 null mutant
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-
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
hemolytic, group A
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
mung bean
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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the mutant called lignescens, a temperature-sensitive mutant that exhibits ectopic lignin deposition and growth defects under high-temperature conditions, is due to single base transition G68S in glucosamine-6-phosphate N-acetyltransferase. When exposed to the restrictive temperature, the mutant strain contains a significantly smaller amount of UDP-GlcNAc than the wild type. The growth defects and ectopic lignification of the mutant are suppressed by the addition of UDP-GlcNAc. N-glycans are reduced and luminal binding protein 3, a typical UPR gene, is expressed in the mutant strain at the restrictive temperature. Treatment with UPR-inducing reagents phenocopies the mutant
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetyl-CoA + D-glucosamine 6-phosphate
CoA + N-acetyl-D-glucosamine 6-phosphate
show the reaction diagram
propionyl-CoA + D-glucosamine 6-phosphate
CoA + N-propionyl-D-glucosamine 6-phosphate
show the reaction diagram
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15% of activity with acetyl-CoA
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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
acetyl-CoA + D-glucosamine 6-phosphate
CoA + N-acetyl-D-glucosamine 6-phosphate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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no cofactor required
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mn2+
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slight but consistent enhancing effect
additional information
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not stimulated by K+, Na+, NH4+ and acetate
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-acetamido-2,6-dideoxy-6-sulfo-D-glucose
2-amino-2,3-dideoxy-3-fluoro-alpha-D-glucopyranosyl phosphate
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inhibition of acetyltransferase activity. Compound cannot substitute the natural substrate GlcN-1-P. Uncompetitive inhibition, compound interacts with the enzymesubstrate complex.
acetyl-CoA
D-Glucosamine-6-phosphate
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feedback inhibition
glucose-6-phosphate
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weak competitive inhibitor
Ni2+
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p-chloromercuribenzoate
p-hydroxymercuribenzoate
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
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activates
free sulfhydryl groups
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activate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.012 - 0.78
acetyl-CoA
0.03 - 7.1
D-Glucosamine 6-phosphate
0.3
D-Glucosamine-6-phosphate
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0.071
glucosamine 6-phosphate
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0.038
Na2S-acetyl coenzyme A
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.12 - 38
D-Glucosamine 6-phosphate
0.076 - 38
glucosamine 6-phosphate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.55
acetyl-CoA
Arabidopsis thaliana
Q9LFU9
pH 7.5, 30C
29
0.52
D-Glucosamine 6-phosphate
Arabidopsis thaliana
Q9LFU9
pH 7.5, 30C
501
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
18.7
2-amino-2,3-dideoxy-3-fluoro-alpha-D-glucopyranosyl phosphate
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pH 7.5, 37C
0.05 - 0.15
acetyl-CoA
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2.6
glucose-6-phosphate
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
12.84
2-amino-2,3-dideoxy-3-fluoro-alpha-D-glucopyranosyl phosphate
Mycobacterium tuberculosis
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pH 7.5, 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00447
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isoenzymes 1 and 2
0.0054
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0.0379
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isoenzyme 3
additional information
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assay method
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
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9.7
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in glycine-NaOH
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8
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approx. 90% of maximal activity at pH 6.0, approx. 95% of maximal activity at pH 8.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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Manually annotated by BRENDA team
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blood-feeding causes no obvious effect on levels of AeGna transcript
Manually annotated by BRENDA team
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mainly expressed in roots, paricularly in the elongation zone of wild-type plants, OsGNA1 transcripts are scarcely detected in roots of mutant NB208
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Brucella abortus (strain 2308)
Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100)
Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
19200
2 * 19200, calculated, 2 * 20000, SDS-PAGE
20000
2 * 19200, calculated, 2 * 20000, SDS-PAGE
21000
x * 21000, SDS-PAGE, immunoblotting, deduced from nucleotide sequence
24000
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25000
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2 * 25000, SDS-PAGE
40300
gel filtration
52000
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x * 52000, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
to 1.5 A resolution. The centre of the protein is a five-stranded beta-sheet, of which four strands are antiparallel-oriented
crystal structures of human and Aspergillus fumigatus GNA1 are determined: structural differences between the two enzymes are mostly located to the sugar-binding site, whereas the AcCoA-binding site is more conserved. These changes affect not only the electrostatics, but also reveal a more spacious sugar binding site in the Aspergillus fumigatus GNA1 enzyme, whereas large side chains at these positions create a tighter pocket in the HsGNA1 enzyme
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using the pseudo-substrate glucose-6-phosphate as a probe with GNA1 crystals, the first GNAT (pseudo-)Michaelis complex is trapped. The complex shows optimal alignment under the ideal Burgi-Dunitz angle for direct nucleophilic attack of the sugar amine, and suggests protonation of the tetrahedral intermediate may proceed through intramolecular proton transfer facilitated by a favourably positioned backbone carbonyl. The hydrogen bond established between the hydroxyl group of Tyr174 and the thioester sulfur increase the electrophilic character of the carbonyl group, favouring the direct nucleophilic attack by the glucosamine 6-phosphate, explaining the 500fold decrease in the turnover when Tyr174 is mutated to Phe, providing direct evidence for the nucleophilic attack of the substrate amine, and giving insight into the protonation of the thiolate leaving group
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crystal structures of HsGNA1 in its apo form, complexed with GlcN6P, and the E156A mutant are solved and refined to 2.7 A, 2.3 A and 2.0 A resolution, respectively. Results reveal new features of the GlcN6P binding in HsGNA1. The conserved charge distribution at the GlcN6P binding pocket is important for the acceptor substrate affinity. Glu156, a conserved residue present in GNA1 from various eukaryotic organisms, plays an important role in both the catalytic reaction and substrate affinity. Moreover, the GlcN6P binds to GNA1 without the help of AcCoA binding, suggesting that a pseudo-substrate could bind GNA1 as an inhibitor without the help of AcCoA
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crystal structures of human and Aspergillus fumigatus GNA1 are determined: structural differences between the two enzymes are mostly located to the sugar-binding site, whereas the AcCoA-binding site is more conserved. These changes affect not only the electrostatics, but also reveal a more spacious sugar binding site in the Aspergillus fumigatus GNA1 enzyme, whereas large side chains at these positions create a tighter pocket in the HsGNA1 enzyme
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hanging-drop vapour-diffusion method. The crystal diffract to better than 2.6 A resolution and belong to space group P4(1)2(1)2 or P4(3)2(1)2. The unit-cell parameters are a = b = 50.08, c = 142.88 A
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to 1.86 A resolution
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
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60C, 10 min, stable
486903
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
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at pH 6, 10 min, stable
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
ampholine or glycine protect against inactivation
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loss of activity with successive freezings and thawings
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, 6 months, several freeze/thaw cycles, no loss of activity
frozen state, 2 weeks, 50% loss of activity
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over liquid N2, more than a month, little loss of activity
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unstable during storage
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
acetone, DEAE-cellulose
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ammonium sulfate, calcium phosphate gel, heat treatment at 65C
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ammonium sulfate, Ultragel AcA54, CoA-Sepharose
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calcium phosphate gel, heat treatment, 60C, mixed bed resin, alumina Cgamma gel, ammonium sulfate
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hydroxylapatite, ammonium sulfate, cellulose phosphate chromatography, electrofocusing, gel electrophoresis
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isoenzymes 1-3, CM-Sephadex
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protamine sulfate, 60C, pH 4.8, mixed-bed resin, ammonium sulfate
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protamine sulfate, pH 4.9, ammonium sulfate, acetone, CM-Sphadex, partially purified
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recombinant His-tagged enzyme
using a HIS-Select HF affinity gel column
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using affinity, ion-exchange and size-exclusion chromatography: yield 25 mg of pure HsGNA1 per litre of bacterial culture
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using affinity, ion-exchange and size-exclusion chromatography: yield 4 mg of pure AfGNA1 per litre of bacterial culture
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
engineering of a metabolic pathway for high level production of N-acetylglucosamine in Escherichia coli by overexpressing Escherichia coli glucosamine synthase (GlmS) and Saccharomyces cerevisiae glucosamine-6-phosphate acetyltransferase (GNA1)
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expressed in Escherichia coli
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expressed in Escherichia coli as a GST-fusion protein
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expressed in Escherichia coli as a His-tagged-fusion protein
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expression in Escherichia coli
expression in Sf9 insect cells and Escherichia coli
metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine by overexpression of glucosamine synthase and glucosamine 6-phosphate N-acetyltransferase and inactivation of catabolic genes
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
enzyme is constitutively transcribed
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G68S
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the mutant called lignescens, a temperature-sensitive mutant that exhibits ectopic lignin deposition and growth defects under high-temperature conditions, is due to single base transition G68S in glucosamine-6-phosphate N-acetyltransferase. When exposed to the restrictive temperature, the mutant strain contains a significantly smaller amount of UDP-GlcNAc than the wild type. The growth defects and ectopic lignification of the mutant are suppressed by the addition of UDP-GlcNAc. N-glycans are reduced and luminal binding protein 3, a typical UPR gene, is expressed in the mutant strain at the restrictive temperature. Treatment with UPR-inducing reagents phenocopies the mutant
E185Y
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Km (mM): 0.03 (D-glucosamine 6-phosphate), 0.04 (acetyl-CoA), kcat (1/sec): 1.34 (D-glucosamine 6-phosphate)
G183E
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Km (mM): 0.056 (D-glucosamine 6-phosphate), 0.1 (acetyl-CoA), kcat (1/sec): 0.34 (D-glucosamine 6-phosphate)
V125R
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Km (mM): 0.6 (D-glucosamine 6-phosphate), 0.2 (acetyl-CoA), kcat (1/sec): 0.12 (D-glucosamine 6-phosphate)
Y174F
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crystal structure of the mutant Y174F acetyl-CoA/glucose-6-P complex is indistinguishable from the wild-type complex, mutant displays some remaining activity, kcat: 0.076/sec (glucosamine 6-phosphate)
Y189R
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Km (mM): 0.056 (D-glucosamine 6-phosphate), 0.086 (acetyl-CoA), kcat (1/sec): 1.7 (D-glucosamine 6-phosphate)
E156A
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Km: 1.2 mM (substrate: glucosamine 6-phosphate), kcat = 9.7/sec (substrate: glucosamine 6-phosphate)
E156D
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Km: 0.175 mM (substrate: glucosamine 6-phosphate), kcat = 19.3/sec (substrate: glucosamine 6-phosphate)
biotechnology
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engineering of a metabolic pathway for high level production of N-acetylglucosamine in Escherichia coli by overexpressing Escherichia coli glucosamine synthase (GlmS) and Saccharomyces cerevisiae glucosamine-6-phosphate acetyltransferase (GNA1)
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
molecular biology