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Information on EC 4.2.1.36 - homoaconitate hydratase
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EC Tree
4.2.1.36 homoaconitate hydrataseIUBMB Comments
Requires a [4Fe-4S] cluster for activity. The enzyme from the hyperthermophilic eubacterium Thermus thermophilus can catalyse the reaction shown above but cannot catalyse the previously described reaction, i.e. formation of (R)-homocitrate by hydration of cis-homoaconitate. The enzyme responsible for the conversion of cis-homoaconitate into (R)-homocitrate in T. thermophilus is unknown at present but the reaction can be catalysed in vitro using aconitate hydratase from pig (EC 4.2.1.3) .
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Word Map
- 4.2.1.36
- alpha-aminoadipate
- homocitrate
- isopropylmalate
-
auxotrophy
- homoisocitrate
- l-lysine
- lys4
- jannaschii
- saccharopine
-
methanocaldococcus
- synthesis
- medicine
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Reaction Schemes
Synonyms
homoaconitase, homoaconitate hydratase, mj1271, 
more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
2-hydroxy-3-carboxyadipate hydro-lyase
-
-
-
-
cis-Homoaconitase
-
-
-
-
HACN
Homoaconitase
Homoaconitate hydratase
-
-
-
-
Hydratase, homoaconitate
-
-
-
-
LYS4
lysF
MJ0499
Homoaconitase
-
-
-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate = (Z)-but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
addition
-
-
-
-
elimination
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
BRENDA
KEGG
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SYSTEMATIC NAME
IUBMB Comments
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate hydro-lyase [(Z)-but-1-ene-1,2,4-tricarboxylate-forming]
Requires a [4Fe-4S] cluster for activity. The enzyme from the hyperthermophilic eubacterium Thermus thermophilus can catalyse the reaction shown above but cannot catalyse the previously described reaction, i.e. formation of (R)-homocitrate by hydration of cis-homoaconitate. The enzyme responsible for the conversion of cis-homoaconitate into (R)-homocitrate in T. thermophilus is unknown at present but the reaction can be catalysed in vitro using aconitate hydratase from pig (EC 4.2.1.3) [2].
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CAS REGISTRY NUMBER
COMMENTARY
9030-68-6
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(Z)-but-1-ene-1,2,4-tricarboxylate + H2O
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
-
i.e. cis-homoaconitate, three different stereoisomeric substrate types, cis-homo1-aconitate, cis-homo2-aconitate, and cis-homo3-aconitate, in the reaction, overview
i.e. homoisocitrate, Arg26 of MJ1271 plays a key role in homoaconitate substrate recognition, while discriminating against the hydrophobic methyl or isopropyl gamma-chains of citraconate and 3-isopropylmalate. Catalytic Ser67 and Arg69 residues in the IMPI small subunit MJ1277 model are equivalent to Ser65 and Arg67 in MJ1271, but residues Val28-Tyr29 replace the polar Arg26-Thr27 residues in the flexible loop region between alpha2 and alpha3
-
r
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
(Z)-but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
-
?
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
(Z)-but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
-
r
2-Hydroxy-3-carboxyadipate
?
-
enzyme is involved in the L-Lys biosynthesis via 2-aminoadipate pathway
-
-
?
2-Hydroxy-3-carboxyadipate
?
-
evaluation of the possible evolutionary scenario for the origin of the Lys biosynthetic pathway by comparison of similarity of homoaconitate hydratase to isopropylmalate isomerase and iron-responsive element binding proteins from fungi and other eukaryotes, eubacteria, and archaea
-
-
?
2-Hydroxy-3-carboxyadipate
?
-
evaluation of the possible evolutionary scenario for the origin of the Lys biosynthetic pathway by comparison of similarity of homoaconitate hydratase to isopropylmalate isomerase and iron-responsive element binding proteins from fungi and other eukaryotes, eubacteria, and archaea
-
-
?
2-Hydroxy-3-carboxyadipate
?
-
enzyme is involved in the L-Lys biosynthesis via 2-aminoadipate pathway
-
-
?
2-Hydroxy-3-carboxyadipate
?
-
enzyme of the homocitrate pathway
-
-
?
2-Hydroxy-3-carboxyadipate
?
-
evaluation of the possible evolutionary scenario for the origin of the Lys biosynthetic pathway by comparison of similarity of homoaconitate hydratase to isopropylmalate isomerase and iron-responsive element binding proteins from fungi and other eukaryotes, eubacteria, and archaea
-
-
?
2-Hydroxy-3-carboxyadipate
?
-
enzyme of the lysine biosynthetic pathway
-
-
?
2-hydroxybutane-1,2,4-tricarboxylate
but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
?
2-hydroxybutane-1,2,4-tricarboxylate
but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
?
2-hydroxybutane-1,2,4-tricarboxylate
but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
?
2-hydroxybutane-1,2,4-tricarboxylate
but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
?
2-hydroxybutane-1,2,4-tricarboxylate
but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
?
2-hydroxybutane-1,2,4-tricarboxylate
but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
?
2-hydroxybutane-1,2,4-tricarboxylate
but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
?
2-hydroxybutane-1,2,4-tricarboxylate
but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
?
additional information
?
-
-
HACNMj is specific for cis-unsaturated tricarboxylates, while isopropylmalate isomerase, IPMIMj, recognizes cis-unsaturated dicarboxylates, substrate specificity determinants of homologous IPMI and HACN proteins from Methanocaldococcus jannaschii from a structural model show characteristic residues in a flexible loop region between R2 and R3 that distinguish HACN from IPMI and aconitase proteins, overview
-
-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
(Z)-but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
-
?
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
(Z)-but-1-ene-1,2,4-tricarboxylate + H2O
-
-
-
-
r
2-Hydroxy-3-carboxyadipate
?
-
enzyme is involved in the L-Lys biosynthesis via 2-aminoadipate pathway
-
-
?
2-Hydroxy-3-carboxyadipate
?
-
evaluation of the possible evolutionary scenario for the origin of the Lys biosynthetic pathway by comparison of similarity of homoaconitate hydratase to isopropylmalate isomerase and iron-responsive element binding proteins from fungi and other eukaryotes, eubacteria, and archaea
-
-
?
2-Hydroxy-3-carboxyadipate
?
-
evaluation of the possible evolutionary scenario for the origin of the Lys biosynthetic pathway by comparison of similarity of homoaconitate hydratase to isopropylmalate isomerase and iron-responsive element binding proteins from fungi and other eukaryotes, eubacteria, and archaea
-
-
?
2-Hydroxy-3-carboxyadipate
?
-
enzyme is involved in the L-Lys biosynthesis via 2-aminoadipate pathway
-
-
?
2-Hydroxy-3-carboxyadipate
?
-
enzyme of the homocitrate pathway
-
-
?
2-Hydroxy-3-carboxyadipate
?
-
evaluation of the possible evolutionary scenario for the origin of the Lys biosynthetic pathway by comparison of similarity of homoaconitate hydratase to isopropylmalate isomerase and iron-responsive element binding proteins from fungi and other eukaryotes, eubacteria, and archaea
-
-
?
2-Hydroxy-3-carboxyadipate
?
-
enzyme of the lysine biosynthetic pathway
-
-
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Zn2+
-
Asp13 and Cys63 side chains from each subunit coordinating Zn2+ ions in small-subunit HACN protein MJ1271
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
trimethyl trans-homoaconitate, trimethyl (1E)1,2-epoxy-butane-1,2,4-tricarboxylate, dimethyl (2S,3R)-2-hydroxy-3-propylsuccinate, dimethyl (2S,3R)-2-hydroxy-3-butylsuccinate, dimethyl (2S,3R)-2-hydroxy-3-allylsuccinate, dimethyl (2R,3S)-2-fluoro-3-propylsuccinate, dimethyl (2R,3S)-2-fluoro-3-butylsuccinate, dimethyl (2R,3S)-2-fluoro-3-allylsuccinate, trimethyl (2R,3S)-2-fluoro-2-deoxyhomoisocitrate, dipotassium (2R,3S)-2-fluoro-3-allylsuccinate, and tripotassium (2R,3S)-2-fluoro-2-deoxyhomoisocitrate have no effect on enzyme activity at concentrations up to 10 mM
-
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DISEASE
TITLE OF PUBLICATION
LINK TO PUBMED
Aspergillosis
Deletion of the Aspergillus fumigatus lysine biosynthesis gene lysF encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis.
Dehydration
Methanogen homoaconitase catalyzes both hydrolyase reactions in coenzyme B biosynthesis.
Infections
Deletion of the Aspergillus fumigatus lysine biosynthesis gene lysF encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis.
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.5
(R)-homocitrate
-
in 50 mM Tris-HCl (pH 8.5), 50 mM KCl, 5 mM MgCl2, 20 mM dithiothreitol, at 20°C
0.022
(Z)-but-1-ene-1,2,4-tricarboxylate
-
wild-type HACNMj, pH 7.0, temperature not specified in the publication
0.135
(Z)-but-1-ene-1,2,4-tricarboxylate
-
mutant R26K, pH 7.0, temperature not specified in the publication
0.22
(Z)-but-1-ene-1,2,4-tricarboxylate
-
mutant R26V, pH 7.0, temperature not specified in the publication
0.22
(Z)-but-1-ene-1,2,4-tricarboxylate
-
mutant T27A, pH 7.0, temperature not specified in the publication
0.3
(Z)-but-1-ene-1,2,4-tricarboxylate
-
pH 7.0, temperature not specified in the publication
0.46
(Z)-but-1-ene-1,2,4-tricarboxylate
-
mutant R26V/T27Y, pH 7.0, temperature not specified in the publication
0.036
(Z)-hex-1-ene-1,2,6-tricarboxylate
-
wild-type HACNMj, pH 7.0, temperature not specified in the publication
0.64
(Z)-hex-1-ene-1,2,6-tricarboxylate
-
mutant R26V/T27Y, pH 7.0, temperature not specified in the publication
0.65
(Z)-hex-1-ene-1,2,6-tricarboxylate
-
mutant T27A, pH 7.0, temperature not specified in the publication
0.66
(Z)-hex-1-ene-1,2,6-tricarboxylate
-
mutant R26V, pH 7.0, temperature not specified in the publication
0.03
(Z)-pent-1-ene-1,2,5-tricarboxylate
-
wild-type HACNMj, pH 7.0, temperature not specified in the publication
0.269
(Z)-pent-1-ene-1,2,5-tricarboxylate
-
mutant T27A, pH 7.0, temperature not specified in the publication
0.87
(Z)-pent-1-ene-1,2,5-tricarboxylate
-
mutant R26V, pH 7.0, temperature not specified in the publication
1.6
(Z)-pent-1-ene-1,2,5-tricarboxylate
-
mutant R26V/T27Y, pH 7.0, temperature not specified in the publication
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.48
(Z)-but-1-ene-1,2,4-tricarboxylate
-
mutant R26V, pH 7.0, temperature not specified in the publication
0.75
(Z)-but-1-ene-1,2,4-tricarboxylate
-
wild-type HACNMj, pH 7.0, temperature not specified in the publication
1.43
(Z)-but-1-ene-1,2,4-tricarboxylate
-
mutant R26K, pH 7.0, temperature not specified in the publication
1.7
(Z)-but-1-ene-1,2,4-tricarboxylate
-
mutant R26V/T27Y, pH 7.0, temperature not specified in the publication
2.5
(Z)-but-1-ene-1,2,4-tricarboxylate
-
mutant T27A, pH 7.0, temperature not specified in the publication
1.9
(Z)-hex-1-ene-1,2,6-tricarboxylate
-
mutant R26V/T27Y, pH 7.0, temperature not specified in the publication
2.5
(Z)-hex-1-ene-1,2,6-tricarboxylate
-
wild-type HACNMj, pH 7.0, temperature not specified in the publication
2.8
(Z)-hex-1-ene-1,2,6-tricarboxylate
-
mutant R26V, pH 7.0, temperature not specified in the publication
4.1
(Z)-hex-1-ene-1,2,6-tricarboxylate
-
mutant T27A, pH 7.0, temperature not specified in the publication
0.66
(Z)-pent-1-ene-1,2,5-tricarboxylate
-
wild-type HACNMj, pH 7.0, temperature not specified in the publication
2.2
(Z)-pent-1-ene-1,2,5-tricarboxylate
-
mutant T27A, pH 7.0, temperature not specified in the publication
5.8
(Z)-pent-1-ene-1,2,5-tricarboxylate
-
mutant R26V, pH 7.0, temperature not specified in the publication
6.6
(Z)-pent-1-ene-1,2,5-tricarboxylate
-
mutant R26V/T27Y, pH 7.0, temperature not specified in the publication
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2.5
(Z)-but-1-ene-1,2,4-tricarboxylate
-
pH 7.0, temperature not specified in the publication
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.58
trans-homoaconitate
Candida albicans
-
in 50 mM Tris-HCl (pH 8.5), 50 mM KCl, 5 mM MgCl2, 20 mM dithiothreitol, at 20°C
5.76
tripotassium (1E)1,2-epoxy-butane-1,2,4-tricarboxylate
Candida albicans
-
in 50 mM Tris-HCl (pH 8.5), 50 mM KCl, 5 mM MgCl2, 20 mM dithiothreitol, at 20°C
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7 - 9
-
pH 7.0: about 35% of maximal activity, pH 9.0: about 95% of maximal activity
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
i.e. Aspergillus nidulans, the mutant strain LysF88 has drastically reduced homoaconitase activity relative to the wild-type strain
-
-
brenda
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
-
the mutations of the small-subunit HACN protein MJ1271 loop-region may reverse the evolution of HACN activity from an ancestral IPMI gene, demonstrating the evolutionary potential for promiscuity in hydro-lyase enzymes. Understanding these specificity determinants enables the functional reannotation of paralogous HACN and isopropylmalate isomerase, IPMI, genes in numerous genome sequences
malfunction
metabolism
-
the enzyme is involved in the lysine biosynthetic pathway
physiological function
additional information
-
the enzymes' stereospecific hydrolyase activity make it an attractive catalyst to produce diastereomers from unsaturated precursors
malfunction
L2 mutant is deficient in homoaconitase activity since it is complemented by the Aspergillus nidulans lysF gene. Wis 54-1255 gene lys3 complements the L2 mutation. Accumulation of homocitric acid in the L2 strain, resulting from the mutation in the lys3 (homoaconitase) gene, is required for the upregulation of the lysine biosynthetic genes. The mutant Lys3 protein is altered in a region required to bind the [4Fe-4S] cluster
malfunction
-
deletion of the LYS4 gene results in lysine auxotrophy. The mutant shows increased sensitivity to oxidative stress, agents that challenge cell wall/membrane integrity, and azole antifungal drugs
physiological function
lysF gene can complement the Penicillium chrysogenum L2 mutant deficient in homoaconitase activity
physiological function
-
homoaconitase proteins catalyze the isomerization of tricarboxylates with variable chain length gamma-carboxylate groups
physiological function
-
Lys4 plays an important role in mitochondrial iron metabolism, and is required for proper mitochondrial function in Cryptococcus neoformans. LYS4 is required for full virulence in Cryptococcus neoformans
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
additional information
-
Methanocaldococcus jannaschii small-subunit HACN protein MJ1271 crystal structure analysis and structural model showing characteristic residues in a flexible loop region between R2 and R3 that distinguish HACN from IPMI and aconitase proteins
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant protein, micro batch method, 18 mg/ml protein in 20 mM Tris-HCl, pH 8.0, containing 200 mM NaCl and 1 mM DTT, is mixed with reservoir solution, containing 50% w/v PEG 200 and 0.1 M Tris-HCl, pH 4.6, at 0.001 ml each and 22°C, X-ray diffraction structure determination and analysis at 2.1 A resolution, molecular replacement method
-
sitting drop vapor diffusion method, small subunit, using 60% (w/v) 2-methyl-2,4-pentanediol and 0.1 M bicine pH 9.7
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
R26K
-
site-directed mutagenesis of small-subunit HACN protein MJ1271, the mutant variant forms a relatively efficient IPMI enzyme
R26V
-
site-directed mutagenesis of small-subunit HACN protein MJ1271, the mutant variant forms a relatively efficient IPMI enzyme. The R26V variant shows detectable dehydratase activity with 3-isopropylmalate
R26V/T27Y
-
site-directed mutagenesis of small-subunit HACN protein MJ1271, the mutant variant resembles the MJ1277 IPMI small subunit in its flexible loop sequence but demonstrates the broad substrate specificity of theR26V variant
T27A
-
site-directed mutagenesis of small-subunit HACN protein MJ1271, the mutant variant has uniformly lower specificity constants for both IPMI and HACN substrates compared to the wild-type enzyme. In a holoenzyme complex, the T27A variant catalyzes the hydration of citraconate and maleate substrates with a 10fold higher KM than wild-type IPMIMj, and the KM values for cis-homoaconitate substrates increase 10-20fold relative to the wild-type HACNMj. The T27A variant has no detectable dehydratase activity with 3-isopropylmalate
additional information
additional information
-
deletion of enzyme gene, results in almost avirulent mutant cells using a low dose intranasal mouse infection model
additional information
-
site-directed mutagenesis of small-subunit HACN protein MJ1271 produces loop-region variant proteins that are reconstituted with wild-type MJ1003 large-subunit protein. The heteromers form promiscuous hydro-lyases with reduced activity but broader substrate specificity, overview
additional information
-
small subunit of enzyme, expression in Thermus thermophilus. Gene product functions by forming a heterodimer with LysT subunit of Thermus thermophilus
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
HisTrap HP column chromatography and Superdex 200 gel filtration
recombinant wild-type and mutant small and large subunit proteins MJ1271 and MJ1003 from Escherichia coli strain BL21(DE3) by heat treatment at 70°C for 10 min, anion and cation exchange chromatography, adsorption chromatography, and gel filtration
-
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli. Introduction of homocitrate synthase, homoaconitase and homoisocitrate dehydrogenase from Saccharomyces cerevisiae into Escherichia coli enables +1 carbon extension from 2-oxoglutarate to 2-oxoadipate, which is subsequently converted into glutarate by a promiscuous 2-oxo acid decarboxylase (KivD) and a succinate semialdehyde dehydrogenase (GabD)
expressioon of wild-type and mutant small and large subunit proteins MJ1271 and MJ1003 in Escherichia coli strain BL21(DE3)
-
lys3 from the parental strain Wis 54-1255 and lys3 mutant allele from strain L2, cloned into pBluescript KS(+)
plasmid pLysF1 expressed in Penicillium chrysogenum L2 mutant strain
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
no LYS4 transcripts are observed in cells grown in low-iron medium, whereas abundant transcripts are observed in the cells grown in the high-iron medium. These results confirm the regulation of LYS4 by iron availability and indicate that the LYS4 gene is mainly transcribed when sufficient iron is present in the environment. Iron availability influences both transcript and protein levels for Lys4
-
transcription factors HAP3 or HAPX negatively regulate expression of the gene, but only in iron-depleted conditions. The transcription factors do not influence LYS4 transcript levels upon iron repletion
-
transcription of the lys3 gene shows a 3fold increase in the transcript level of this gene in strain L2 as compared with the parental strain Wis 54-1255
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
-
deletion of enzyme gene, results in almost avirulent mutant cells using a low dose intranasal mouse infection model
synthesis
synthesis
-
the enzymes' stereospecific hydrolyase activity make it an attractive catalyst to produce diastereomers from unsaturated precursors, analysis of the structural basis for engineering of new stereospecific hydro-lyase enzymes for chemoenzymatic syntheses, overview
synthesis
glutarate biosynthetic pathway by incorporation of a +1 carbon chain extension pathway from 2-oxoglutarate in combination with 2-oxo acid decarboxylation pathway in Escherichia coli. Introduction of homocitrate synthase, homoaconitase and homoisocitrate dehydrogenase from Saccharomyces cerevisiae into Escherichia coli enables +1 carbon extension from 2-oxoglutarate to 2-oxoadipate, which is subsequently converted into glutarate by a promiscuous 2-oxo acid decarboxylase (KivD) and a succinate semialdehyde dehydrogenase (GabD). The recombinant Escherichia coli coexpressing all five genes produces 0.3 g/l glutarate from glucose. To further improve the titers, 2-oxoglutarate is rechanneled into carbon chain extension pathway via the clustered regularly interspersed palindromic repeats system mediated interference (CRISPRi) of essential genes sucA and sucB in tricarboxylic acid cycle. The final strain can produce 0.42 g/l glutarate, which is increased by 40% compared with the parental strain. Glutarate is one of the most potential building blocks for bioplastics
synthesis
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glutarate biosynthetic pathway by incorporation of a +1 carbon chain extension pathway from 2-oxoglutarate in combination with 2-oxo acid decarboxylation pathway in Escherichia coli. Introduction of homocitrate synthase, homoaconitase and homoisocitrate dehydrogenase from Saccharomyces cerevisiae into Escherichia coli enables +1 carbon extension from 2-oxoglutarate to 2-oxoadipate, which is subsequently converted into glutarate by a promiscuous 2-oxo acid decarboxylase (KivD) and a succinate semialdehyde dehydrogenase (GabD). The recombinant Escherichia coli coexpressing all five genes produces 0.3 g/l glutarate from glucose. To further improve the titers, 2-oxoglutarate is rechanneled into carbon chain extension pathway via the clustered regularly interspersed palindromic repeats system mediated interference (CRISPRi) of essential genes sucA and sucB in tricarboxylic acid cycle. The final strain can produce 0.42 g/l glutarate, which is increased by 40% compared with the parental strain. Glutarate is one of the most potential building blocks for bioplastics
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