Information on EC 4.2.1.36 - homoaconitate hydratase

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The expected taxonomic range for this enzyme is: Eukaryota, Archaea, Bacteria

EC NUMBER
COMMENTARY hide
4.2.1.36
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RECOMMENDED NAME
GeneOntology No.
homoaconitate hydratase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate = (Z)-but-1-ene-1,2,4-tricarboxylate + H2O
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
addition
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elimination
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
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coenzyme B biosynthesis
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L-lysine biosynthesis IV
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L-lysine biosynthesis V
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Lysine biosynthesis
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Metabolic pathways
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Microbial metabolism in diverse environments
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NIL
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lysine metabolism
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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].
CAS REGISTRY NUMBER
COMMENTARY hide
9030-68-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
strain Wis 54-1255 and strain L2
UniProt
Manually annotated by BRENDA team
small subunit of enzyme, expression in Thermus thermophilus
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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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
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
physiological function
additional information
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the enzymes' stereospecific hydrolyase activity make it an attractive catalyst to produce diastereomers from unsaturated precursors
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
(Z)-but-1-ene-1,2,4-tricarboxylate + H2O
show the reaction diagram
(R)-homocitrate
?
show the reaction diagram
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?
(Z)-but-1-ene-1,2,4-tricarboxylate + H2O
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
show the reaction diagram
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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
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r
(Z)-but-1-ene-1,2,4-tricarboxylic acid
?
show the reaction diagram
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?
(Z)-hex-1-ene-1,2,6-tricarboxylate
?
show the reaction diagram
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?
(Z)-pent-1-ene-1,2,5-tricarboxylate
?
show the reaction diagram
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?
2-Hydroxy-3-carboxyadipate
?
show the reaction diagram
2-hydroxybutane-1,2,4-tricarboxylate
but-1-ene-1,2,4-tricarboxylate + H2O
show the reaction diagram
cis-homoaconitate
homoisocitrate + H2O
show the reaction diagram
cis-homoaconitate + H2O
(2R,3S)-homoisocitrate
show the reaction diagram
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?
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
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
(Z)-but-1-ene-1,2,4-tricarboxylate + H2O
show the reaction diagram
2-Hydroxy-3-carboxyadipate
?
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Zn2+
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Asp13 and Cys63 side chains from each subunit coordinating Zn2+ ions in small-subunit HACN protein MJ1271
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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2,2'-dipyridyl
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p-chloromercuribenzoate
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trans-homoaconitate
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tripotassium (1E)1,2-epoxy-butane-1,2,4-tricarboxylate
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.5
(R)-homocitrate
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in 50 mM Tris-HCl (pH 8.5), 50 mM KCl, 5 mM MgCl2, 20 mM dithiothreitol, at 20°C
0.022 - 0.46
(Z)-but-1-ene-1,2,4-tricarboxylate
0.036 - 0.66
(Z)-hex-1-ene-1,2,6-tricarboxylate
0.03 - 1.6
(Z)-pent-1-ene-1,2,5-tricarboxylate
1.1
2-hydroxy-3-carboxyadipate
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.48 - 2.5
(Z)-but-1-ene-1,2,4-tricarboxylate
1.9 - 4.1
(Z)-hex-1-ene-1,2,6-tricarboxylate
0.66 - 6.6
(Z)-pent-1-ene-1,2,5-tricarboxylate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.5
(Z)-but-1-ene-1,2,4-tricarboxylate
Methanocaldococcus jannaschii
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pH 7.0, temperature not specified in the publication
4396
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.58
trans-homoaconitate
Candida albicans
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in 50 mM Tris-HCl (pH 8.5), 50 mM KCl, 5 mM MgCl2, 20 mM dithiothreitol, at 20°C
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5.76
tripotassium (1E)1,2-epoxy-butane-1,2,4-tricarboxylate
Candida albicans
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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 hide
LITERATURE
8.1 - 8.6
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potassium phosphate buffer
8.6
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phosphate buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9
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pH 7.0: about 35% of maximal activity, pH 9.0: about 95% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
84490
Wis 54-1255 gene lys3, sequence analysis
143000
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recombinant wild-type HACN, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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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
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
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sitting drop vapor diffusion method, small subunit, using 60% (w/v) 2-methyl-2,4-pentanediol and 0.1 M bicine pH 9.7
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
expressed in Escherichia coli BL21(DE3) Star cells
expressioon of wild-type and mutant small and large subunit proteins MJ1271 and MJ1003 in Escherichia coli strain BL21(DE3)
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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
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
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
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R26K
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site-directed mutagenesis of small-subunit HACN protein MJ1271, the mutant variant forms a relatively efficient IPMI enzyme
R26V
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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
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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
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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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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deletion of enzyme gene, results in almost avirulent mutant cells using a low dose intranasal mouse infection model
synthesis
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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
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