Information on EC 4.2.1.114 - methanogen homoaconitase

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The expected taxonomic range for this enzyme is: Methanocaldococcus jannaschii

EC NUMBER
COMMENTARY hide
4.2.1.114
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RECOMMENDED NAME
GeneOntology No.
methanogen homoaconitase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(R)-2-hydroxybutane-1,2,4-tricarboxylate = (1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
show the reaction diagram
overall reaction
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(R)-2-hydroxybutane-1,2,4-tricarboxylate = (Z)-but-1-ene-1,2,4-tricarboxylate + H2O
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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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
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coenzyme B biosynthesis
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Lysine biosynthesis
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Metabolic pathways
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Methane metabolism
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Microbial metabolism in diverse environments
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lysine metabolism
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SYSTEMATIC NAME
IUBMB Comments
(R)-2-hydroxybutane-1,2,4-tricarboxylate hydro-lyase [(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate-forming]
This enzyme catalyses several reactions in the pathway of coenzyme-B biosynthesis in methanogenic archaea. Requires a [4Fe-4S] cluster for activity. In contrast to EC 4.2.1.36, homoaconitate hydratase, this enzyme can catalyse both the dehydration of (R)-homocitrate to form cis-homoaconitate and the subsequent hydration reaction that forms homoisocitrate. In addition to cis-homoaconitate, the enzyme can also catalyse the hydration of the physiological substrates dihomocitrate and trihomocitrate as well as the non-physiological substrate tetrahomocitrate. cis-Aconitate and threo-DL-isocitrate cannot act as substrates, and (S)-homocitrate and trans-homoaconitate act as inhibitors of the enzyme.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
large subunit of homoaconitase
SwissProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(1E)-hex-1-ene-1,2,6-tricarboxylic acid + H2O
(2R)-hexane-1,2,6-tricarboxyclic acid
show the reaction diagram
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?
(1E)-pent-1-ene-1,2,5-tricarboxylic acid + H2O
(2R)-pentane-1,2,5-tricarboxylic acid
show the reaction diagram
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?
(2R)-butane-1,2,4-tricarboxylic acid
but-1-ene-1,2,4-tricarboxylic acid + H2O
show the reaction diagram
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?
(R)-2-hydroxybutane-1,2,4-tricarboxylate
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
show the reaction diagram
i.e. (R)-homocitrate, overall reaction, the enzyme catalyzes both the dehydration of (R)-homocitrate to form cis-homoaconitate, and hydration producing homoisocitrate
i.e. homoisocitrate
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r
(R)-2-hydroxybutane-1,2,4-tricarboxylate
(Z)-but-1-ene-1,2,4-tricarboxylate + H2O
show the reaction diagram
i.e. (R)-homocitrate, first half-reaction
i.e. cis-homoaconitate, the activity in the reverse direction is similar as with (R)-homocitrate
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r
(Z)-but-1-ene-1,2,4-tricarboxylate + H2O
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
show the reaction diagram
second half-reaction
i.e. homoisocitrate
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r
but-1-ene-1,2,4-tricarboxylic acid + H2O
(2R)-butane-1,2,4-tricarboxylic acid
show the reaction diagram
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?
cis-(homo)2aconitate + H2O
(-)-threo-iso(homo)2citrate
show the reaction diagram
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?
cis-(homo)2aconitate + H2O
?
show the reaction diagram
the activity is similar as with (R)-homocitrate
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?
cis-(homo)3aconitate + H2O
(-)-threo-iso(homo)3citrate
show the reaction diagram
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?
cis-(homo)3aconitate + H2O
?
show the reaction diagram
the activity is similar as with (R)-homocitrate
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?
cis-(homo)4aconitate + H2O
?
show the reaction diagram
the activity is similar as with (R)-homocitrate
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?
dihomocitrate
cis-(homo)2aconitate + H2O
show the reaction diagram
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?
maleate + H2O
?
show the reaction diagram
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?
trihomocitrate
cis-(homo)3aconitate + H2O
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
cis-(homo)2aconitate + H2O
(-)-threo-iso(homo)2citrate
show the reaction diagram
Q58991
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?
cis-(homo)3aconitate + H2O
(-)-threo-iso(homo)3citrate
show the reaction diagram
Q58991
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?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
the enzyme contains an iron-sulfur center, reconstitution of the purified recombinant apoenzyme with iron-sulfur, overview
Iron-sulfur cluster
the structure of the protein has unbound Fe-S clusters and contains a fourth cysteine in the active site
Mg2+
required for the hydratase reaction, not for the hydrolase reaction
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(S)-homocitrate
complete inhibition at 0.1 mM
cis-aconitate
50% inhibition of the hydrolase activity at 0.025 mM
cis-homoaconitate/(R)-homocitrate
mixtures of 0.1 mM cis-homoaconitate and 0.05 mM (R)-homocitrate reduce the HACN hydrolyase activity by 50%
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trans-(homo)3aconitate
50% inhibition of the hydrolase activity at 0.4 mM
trans-aconitate
50% inhibition of the hydrolase activity at 0.1 mM
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.036 - 0.66
(1E)-hex-1-ene-1,2,6-tricarboxylic acid
0.03 - 1.6
(1E)-pent-1-ene-1,2,5-tricarboxylic acid
1.5
(R)-homocitrate
pH 8.5, 60°C, recombinant enzyme, dehydratase reaction
0.022 - 0.46
but-1-ene-1,2,4-tricarboxylic acid
0.03
cis-homo2aconitate
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
0.036
cis-homo3aconitate
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
0.175
cis-homo4aconitate
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
0.022
cis-homoaconitate
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
0.33
Maleate
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.9 - 4.1
(1E)-hex-1-ene-1,2,6-tricarboxylic acid
0.66 - 6.6
(1E)-pent-1-ene-1,2,5-tricarboxylic acid
0.37
(R)-homocitrate
Methanocaldococcus jannaschii
Q58991
pH 8.5, 60°C, recombinant enzyme, dehydratase reaction
0.48 - 2.5
but-1-ene-1,2,4-tricarboxylic acid
0.66
cis-homo2aconitate
Methanocaldococcus jannaschii
Q58991
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
2.5
cis-homo3aconitate
Methanocaldococcus jannaschii
Q58991
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
5.6
cis-homo4aconitate
Methanocaldococcus jannaschii
Q58991
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
0.75
cis-homoaconitate
Methanocaldococcus jannaschii
Q58991
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
6
Maleate
Methanocaldococcus jannaschii
Q58991
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
hydrolase and hydratase assay at
PDB
SCOP
CATH
ORGANISM
UNIPROT
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
heterotetramer, MJ1003 and MJ1271 proteins form an active homoaconitase enzyme
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting-drop vapour-diffusion method and hanging-drop vapour diffusion at 20°C, structures of the large subunit of homoaconise is reported at 2.5 A resolution
small-subunit HACN protein (MJ1271), micro batch method, using 50% (w/v) polyethylene glycol 200 and 0.1 M Tris-HCl, pH 4.6, at 20°C
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant MJ1003 and MJ1271 proteins from Escherichia coli strain BL21(DE3) by heat treatment of the cell lysate, followed by anion exchange chromatography, dialysis, and ultrafiltration
Toyopearl SuperQ-650 M column chromatography, Resource S column chromatography, and Superdex 200 gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, coexpression of MJ1003 and MJ1271 proteins in Escherichia coli strain BL21(DE3)
expression in Escherichia coli BL21(DE3) Star cells
small-subunit HACN protein MJ1271 is expressed in Escherichia coli BL21 CodonPlus (DE3)-RIL cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R26K
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the variant forms an relatively efficient isopropylmalate isomerase enzyme
R26V
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the variant forms an relatively efficient isopropylmalate isomerase enzyme
R26V/T27Y
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the variant resembles the MJ1277 isopropylmalate isomerase small subunit in its flexible loop sequence but demonstrates the broad substrate specificity of the R26V variant
T27A t
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the variant has uniformly lower specificity constants for both isopropylmalate isomerase and methanogen homoaconitase substrates
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