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Information on EC 4.2.1.114 - methanogen homoaconitase and Organism(s) Methanocaldococcus jannaschii and UniProt Accession Q58409
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4.2.1.114 methanogen homoaconitaseIUBMB Comments
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 dihomoaconitate and trihomoaconitate as well as the non-physiological substrate tetrahomoaconitate. cis-Aconitate and threo-DL-isocitrate cannot act as substrates, and (S)-homocitrate and trans-homoaconitate act as inhibitors of the enzyme.
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The taxonomic range for the selected organisms is: Methanocaldococcus jannaschii
The expected taxonomic range for this enzyme is: Archaea, Bacteria
The expected taxonomic range for this enzyme is: Archaea, Bacteria
Reaction Schemes
Synonyms
mj1003, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
HACN
methanogen HACN
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-
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additional information
the enzyme belongs to the aconitase superfamily
PATHWAY SOURCE
PATHWAYS
BRENDA
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 dihomoaconitate and trihomoaconitate as well as the non-physiological substrate tetrahomoaconitate. cis-Aconitate and threo-DL-isocitrate cannot act as substrates, and (S)-homocitrate and trans-homoaconitate act as inhibitors of the enzyme.
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
(1E)-hex-1-ene-1,2,6-tricarboxylic acid + H2O
(2R)-hexane-1,2,6-tricarboxyclic acid
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-
-
-
?
(1E)-pent-1-ene-1,2,5-tricarboxylic acid + H2O
(2R)-pentane-1,2,5-tricarboxylic acid
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-
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?
(2R)-butane-1,2,4-tricarboxylic acid
but-1-ene-1,2,4-tricarboxylic acid + H2O
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-
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?
(R)-2-hydroxybutane-1,2,4-tricarboxylate
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
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
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
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
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?
cis-(homo)2aconitate + H2O
(-)-threo-iso(homo)2citrate
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?
cis-(homo)2aconitate + H2O
?
the activity is similar as with (R)-homocitrate
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?
cis-(homo)3aconitate + H2O
(-)-threo-iso(homo)3citrate
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?
cis-(homo)3aconitate + H2O
?
the activity is similar as with (R)-homocitrate
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-
?
cis-(homo)4aconitate + H2O
?
the activity is similar as with (R)-homocitrate
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?
additional information
?
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homoaconitase enzymes catalyze hydrolyase reactions in the alpha-aminoadipate pathway for lysine biosynthesis or the 2-oxosuberate pathway for methanogenic coenzyme B biosynthesis
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?
additional information
?
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homoaconitase enzymes catalyze hydrolyase reactions in the alpha-aminoadipate pathway for lysine biosynthesis or the 2-oxosuberate pathway for methanogenic coenzyme B biosynthesis
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?
additional information
?
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homoaconitase together with the endogenous homoisocitrate dehydrogenase, EC 1.1.1.87, catalyze all of the isomerization and oxidative decarboxylation reactions required to form 2-oxoadipate, 2-oxopimelate, and 2-oxosuberate, completing three iterations of the 2-oxoacid elongation pathway. Methanogenic archaeal homoaconitases and fungal homoaconitases evolved in parallel in the aconitase superfamily, LC-MS analysis of enzyme reaction products, overview
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additional information
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homoaconitase together with the endogenous homoisocitrate dehydrogenase, EC 1.1.1.87, catalyze all of the isomerization and oxidative decarboxylation reactions required to form 2-oxoadipate, 2-oxopimelate, and 2-oxosuberate, completing three iterations of the 2-oxoacid elongation pathway. Methanogenic archaeal homoaconitases and fungal homoaconitases evolved in parallel in the aconitase superfamily, LC-MS analysis of enzyme reaction products, overview
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?
additional information
?
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HACN has no detectable activity with citraconate or isopropylmalate
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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
cis-(homo)2aconitate + H2O
(-)-threo-iso(homo)2citrate
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-
-
?
cis-(homo)3aconitate + H2O
(-)-threo-iso(homo)3citrate
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-
-
?
additional information
?
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-
homoaconitase enzymes catalyze hydrolyase reactions in the alpha-aminoadipate pathway for lysine biosynthesis or the 2-oxosuberate pathway for methanogenic coenzyme B biosynthesis
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-
?
additional information
?
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homoaconitase enzymes catalyze hydrolyase reactions in the alpha-aminoadipate pathway for lysine biosynthesis or the 2-oxosuberate pathway for methanogenic coenzyme B biosynthesis
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?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Iron-sulfur cluster
the structure of the protein has unbound Fe-S clusters and contains a fourth cysteine in the active site
Fe2+
the enzyme contains an iron-sulfur center, reconstitution of the purified recombinant apoenzyme with iron-sulfur, overview
Mg2+
required for the hydratase reaction, not for the hydrolase reaction
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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%
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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no inhibition by citraconate up to 0.2 mM
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additional information
no inhibition by citraconate up to 0.2 mM
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.5
(R)-homocitrate
pH 8.5, 60°C, recombinant enzyme, dehydratase reaction
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
0.036
(1E)-hex-1-ene-1,2,6-tricarboxylic acid
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wild type enzyme
0.64
(1E)-hex-1-ene-1,2,6-tricarboxylic acid
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mutant enzyme R26V/T27Y
0.65
(1E)-hex-1-ene-1,2,6-tricarboxylic acid
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mutant enzyme T27A
0.66
(1E)-hex-1-ene-1,2,6-tricarboxylic acid
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mutant enzyme R26V
0.03
(1E)-pent-1-ene-1,2,5-tricarboxylic acid
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wild type enzyme
0.269
(1E)-pent-1-ene-1,2,5-tricarboxylic acid
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mutant enzyme T27A
0.87
(1E)-pent-1-ene-1,2,5-tricarboxylic acid
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mutant enzyme R26V
1.6
(1E)-pent-1-ene-1,2,5-tricarboxylic acid
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mutant enzyme R26V/T27Y
0.46
but-1-ene-1,2,4-tricarboxylic acid
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mutant enzyme R26V/T27Y
additional information
additional information
-
steady-state kinetics
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additional information
additional information
steady-state kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.37
(R)-homocitrate
pH 8.5, 60°C, recombinant enzyme, dehydratase reaction
0.66
cis-homo2aconitate
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
2.5
cis-homo3aconitate
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
5.6
cis-homo4aconitate
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
0.75
cis-homoaconitate
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
6
Maleate
pH 9.0, 60°C, recombinant enzyme, hydrolase reaction
1.9
(1E)-hex-1-ene-1,2,6-tricarboxylic acid
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mutant enzyme R26V/T27Y
2.8
(1E)-hex-1-ene-1,2,6-tricarboxylic acid
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mutant enzyme R26V
4.1
(1E)-hex-1-ene-1,2,6-tricarboxylic acid
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mutant enzyme T27A
0.66
(1E)-pent-1-ene-1,2,5-tricarboxylic acid
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wild type enzyme
2.2
(1E)-pent-1-ene-1,2,5-tricarboxylic acid
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mutant enzyme T27A
5.8
(1E)-pent-1-ene-1,2,5-tricarboxylic acid
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mutant enzyme R26V
6.6
(1E)-pent-1-ene-1,2,5-tricarboxylic acid
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mutant enzyme R26V/T27Y
1.7
but-1-ene-1,2,4-tricarboxylic acid
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mutant enzyme R26V/T27Y
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
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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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
R26K
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the variant forms an relatively efficient isopropylmalate isomerase enzyme
R26V
-
the variant forms an relatively efficient isopropylmalate isomerase enzyme
R26V/T27Y
-
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
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)
small-subunit HACN protein MJ1271 is expressed in Escherichia coli BL21 CodonPlus (DE3)-RIL cells
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Drevland, R.M.; Jia, Y.; Palmer, D.R.; Graham, D.E.
Methanogen homoaconitase catalyzes both hydrolyase reactions in coenzyme B biosynthesis
J. Biol. Chem.
283
28888-28896
2008
Methanocaldococcus jannaschii, Methanocaldococcus jannaschii (Q58991)
Jeyakanthan, J.; Drevland, R.M.; Gayathri, D.R.; Velmurugan, D.; Shinkai, A.; Kuramitsu, S.; Yokoyama, S.; Graham, D.E.
Substrate specificity determinants of the methanogen homoaconitase enzyme: structure and function of the small subunit
Biochemistry
49
2687-2696
2010
Methanocaldococcus jannaschii
Lee, E.H.; Lee, K.; Hwang, K.Y.
Structural characterization and comparison of the large subunits of IPM isomerase and homoaconitase from Methanococcus jannaschii
Acta Crystallogr. Sect. D
70
922-931
2014
Methanocaldococcus jannaschii (Q58409), Methanocaldococcus jannaschii, Methanocaldococcus jannaschii DSM 2661 (Q58409)
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