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Literature summary extracted from

  • Borjian, F.; Johnsen, U.; Schoenheit, P.; Berg, I.A.
    Succinyl-CoA mesaconate CoA-transferase and mesaconyl-CoA hydratase, enzymes of the methylaspartate cycle in Haloarcula hispanica (2017), Front. Microbiol., 8, 1683 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.8.3.26 Haloferax volcanii strain H1209 is used for the heterologous expression of the Haloarcula hispanica proteins Haloarcula hispanica
4.2.1.148 gene Hah_1340, phylogenetic analysis, recombinant expression of soluble His-tagged enzyme in Haloferax volcanii strain H1209 Haloarcula hispanica
4.2.1.148 Haloferax volcanii strain H1209 is used for the heterologous expression of the Haloarcula hispanica proteins Haloarcula hispanica

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.8.3.26 CaCl2 5 mM, 5% inhibition Haloarcula hispanica
2.8.3.26 CoCl2 5 mM, 10% inhibition Haloarcula hispanica
2.8.3.26 MgCl2 5 mM, 7% inhibition Haloarcula hispanica
2.8.3.26 MnCl2 5 mM, 24% inhibition Haloarcula hispanica
2.8.3.26 additional information no inhibition by EDTA Haloarcula hispanica
2.8.3.26 NiCl2 5 mM, 5% inhibition Haloarcula hispanica
4.2.1.148 CaCl2 18% inhibition at 5 mM; 5 mM, 18% inhibition Haloarcula hispanica
4.2.1.148 CoCl2 40% inhibition at 5 mM; 5 mM, 34% inhibition Haloarcula hispanica
4.2.1.148 MgCl2 11% inhibition at 5 mM; 5 mM, 11% inhibition Haloarcula hispanica
4.2.1.148 MnCl2 22% inhibition at 5 mM; 5 mM, 22% inhibition Haloarcula hispanica
4.2.1.148 additional information no inhibition by EDTA; no inhibition by EDTA at 2 mM Haloarcula hispanica
4.2.1.148 NiCl2 34% inhibition at 5 mM; 5 mM, 40% inhibition Haloarcula hispanica

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.8.3.26 0.6
-
Methylsuccinate 37°C, pH 7.8, 1 mM succinyl-CoA Haloarcula hispanica
2.8.3.26 1.3
-
mesaconate 37°C, pH 7.8, 1 mM succinyl-CoA Haloarcula hispanica
2.8.3.26 2.8
-
succinyl-CoA 37°C, pH 7.8 Haloarcula hispanica
2.8.3.26 2.9
-
Methylsuccinate 37°C, pH 7.8, 5 mM succinyl-CoA Haloarcula hispanica
2.8.3.26 7.1
-
mesaconate 37°C, pH 7.8, 5 mM succinyl-CoA Haloarcula hispanica
2.8.3.26 39.3
-
Acrylate 37°C, pH 7.8 Haloarcula hispanica
2.8.3.26 40.9
-
Glutarate 37°C, pH 7.8 Haloarcula hispanica
4.2.1.148 0.18
-
3-methylfumaryl-CoA 37°C, pH 7.8 Haloarcula hispanica
4.2.1.148 0.32
-
(S)-malyl-CoA 37°C, pH 7.8 Haloarcula hispanica
4.2.1.148 0.35
-
(2R,3S)-2-methylmalyl-CoA 37°C, pH 7.8 Haloarcula hispanica
4.2.1.148 1
-
2-methylfumaryl-CoA 37°C, pH 7.8 Haloarcula hispanica
4.2.1.148 2.6
-
(S)-citramalyl-CoA 37°C, pH 7.8 Haloarcula hispanica

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.8.3.26 KCl the enzyme is dependent on the presence of KCl and shows highest activity at 4 M KCl Haloarcula hispanica
2.8.3.26 additional information no requirement for divalent cations Haloarcula hispanica
4.2.1.148 KCl the enzyme is dependent on the presence of KCl and shows highest activity at 0.5 M KCl. With 82% of activity at 4 M KCl, whereas the activity dropps severely at 0.1 M KCl Haloarcula hispanica
4.2.1.148 additional information no requirement for divalent cations Haloarcula hispanica

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.8.3.26 71000
-
gel filtration Haloarcula hispanica
4.2.1.148 86000
-
gel filtration Haloarcula hispanica
4.2.1.148 86000
-
recombinant His-tagged enzyme, gel filtration Haloarcula hispanica

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.8.3.26 mesaconate + succinyl-CoA Haloarcula hispanica the enzyme is involved in the methylaspartate cycle 2-methylfumaryl-CoA + succinate
-
?
4.2.1.148 2-methylfumaryl-CoA + H2O Haloarcula hispanica i.e. mesaconyl-CoA. The enzyme catalyzes a reaction in the methylaspartate cycle (2R,3S)-2-methylmalyl-CoA
-
?
4.2.1.148 2-methylfumaryl-CoA + H2O Haloarcula hispanica ATCC 33960 / DSM 4426 / JCM 8911 / NBRC 102182 / NCIMB 2187 / VKM B-1755 i.e. mesaconyl-CoA. The enzyme catalyzes a reaction in the methylaspartate cycle (2R,3S)-2-methylmalyl-CoA
-
?
4.2.1.148 2-methylfumaryl-CoA + H2O Haloarcula hispanica DSM 4426 i.e. mesaconyl-CoA. The enzyme catalyzes a reaction in the methylaspartate cycle (2R,3S)-2-methylmalyl-CoA
-
?
4.2.1.148 mesaconyl-C1-CoA Haloarcula hispanica the physiological substrate is mesaconyl-C1-CoA (2R,3S)-2-methyl-3-hydroxysuccinyl-C1-CoA + H2O
-
r
4.2.1.148 mesaconyl-C1-CoA Haloarcula hispanica ATCC 33960 / DSM 4426 / JCM 8911 / NBRC 102182 / NCIMB 2187 / VKM B-1755 the physiological substrate is mesaconyl-C1-CoA (2R,3S)-2-methyl-3-hydroxysuccinyl-C1-CoA + H2O
-
r
4.2.1.148 mesaconyl-C1-CoA Haloarcula hispanica DSM 4426 the physiological substrate is mesaconyl-C1-CoA (2R,3S)-2-methyl-3-hydroxysuccinyl-C1-CoA + H2O
-
r

Organism

EC Number Organism UniProt Comment Textmining
2.8.3.26 Haloarcula hispanica G0HQ31
-
-
4.2.1.148 Haloarcula hispanica G0HQ35
-
-
4.2.1.148 Haloarcula hispanica ATCC 33960 / DSM 4426 / JCM 8911 / NBRC 102182 / NCIMB 2187 / VKM B-1755 G0HQ35
-
-
4.2.1.148 Haloarcula hispanica DSM 4426 G0HQ35
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.8.3.26
-
Haloarcula hispanica
4.2.1.148
-
Haloarcula hispanica
4.2.1.148 recombinant His-tagged enzyme 427fold from Haloferax volcanii strain H1209 by nickel affinity chromatography and gel filtration Haloarcula hispanica

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.8.3.26 16.9
-
37°C, pH 7.8 Haloarcula hispanica
4.2.1.148 157
-
37°C, pH 7.8 Haloarcula hispanica
4.2.1.148 157
-
purified recombinant His-tagged enzyme, substrate mesaconyl-C1-CoA, pH 7.8, 37°C Haloarcula hispanica

Storage Stability

EC Number Storage Stability Organism
4.2.1.148 -20°C, purified recombinant enzyme, 50 mM Tris/HCl, pH 8.0, 2 M KCl, 3 weeks, complete loss of activity Haloarcula hispanica
4.2.1.148 4°C, purified recombinant enzyme, 50 mM Tris/HCl, pH 8.0, 2 M KCl, 3 weeks, loss of 70-80% activity Haloarcula hispanica

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.8.3.26 acrylate + succinyl-CoA low activity Haloarcula hispanica ? + succinate
-
?
2.8.3.26 glutarate + succinyl-CoA low activity Haloarcula hispanica ? + succinate
-
?
2.8.3.26 itaconate + succinyl-CoA low activity Haloarcula hispanica ? + succinate
-
?
2.8.3.26 mesaconate + succinyl-CoA the enzyme is involved in the methylaspartate cycle Haloarcula hispanica 2-methylfumaryl-CoA + succinate
-
?
2.8.3.26 mesaconate + succinyl-CoA whereas the enzyme is highly specific for succinyl-CoA as a CoA-donor, it can activate other carboxylic acids, i.e., methylsuccinate, itaconate, acrylate, and glutarate Haloarcula hispanica 2-methylfumaryl-CoA + succinate
-
?
2.8.3.26 methylsuccinate + succinyl-CoA high activity, not a natural substrate Haloarcula hispanica ? + succinate
-
?
4.2.1.148 (2R,3S)-2-methyl-3-hydroxysuccinyl-C4-CoA + H2O i.e. (S)-citramalyl-CoA Haloarcula hispanica 3-methylfumaryl-CoA + H2O
-
r
4.2.1.148 (2R,3S)-2-methylmalyl-CoA the equilibrium of the reaction is on the side of (2R,3S)-2-methylmalyl-CoA Haloarcula hispanica 2-methylfumaryl-CoA + H2O
-
r
4.2.1.148 (2R,3S)-2-methylmalyl-CoA the equilibrium of the reaction is on the side of (2R,3S)-2-methylmalyl-CoA Haloarcula hispanica ATCC 33960 / DSM 4426 / JCM 8911 / NBRC 102182 / NCIMB 2187 / VKM B-1755 2-methylfumaryl-CoA + H2O
-
r
4.2.1.148 (2R,3S)-2-methylmalyl-CoA the equilibrium of the reaction is on the side of (2R,3S)-2-methylmalyl-CoA Haloarcula hispanica DSM 4426 2-methylfumaryl-CoA + H2O
-
r
4.2.1.148 (S)-citramalyl-CoA
-
Haloarcula hispanica 3-methylfumaryl-CoA + H2O
-
r
4.2.1.148 (S)-malyl-CoA
-
Haloarcula hispanica fumaryl-CoA + H2O
-
r
4.2.1.148 2-methylfumaryl-CoA + H2O i.e. mesaconyl-CoA. The enzyme catalyzes a reaction in the methylaspartate cycle Haloarcula hispanica (2R,3S)-2-methylmalyl-CoA
-
?
4.2.1.148 2-methylfumaryl-CoA + H2O i.e. mesaconyl-CoA, i.e. mesaconyl-C1-CoA. The equilibrium of the reaction is on the side of (2R,3S)-2-methylmalyl-CoA Haloarcula hispanica (2R,3S)-2-methylmalyl-CoA
-
r
4.2.1.148 2-methylfumaryl-CoA + H2O i.e. mesaconyl-CoA. The enzyme catalyzes a reaction in the methylaspartate cycle Haloarcula hispanica ATCC 33960 / DSM 4426 / JCM 8911 / NBRC 102182 / NCIMB 2187 / VKM B-1755 (2R,3S)-2-methylmalyl-CoA
-
?
4.2.1.148 2-methylfumaryl-CoA + H2O i.e. mesaconyl-CoA, i.e. mesaconyl-C1-CoA. The equilibrium of the reaction is on the side of (2R,3S)-2-methylmalyl-CoA Haloarcula hispanica ATCC 33960 / DSM 4426 / JCM 8911 / NBRC 102182 / NCIMB 2187 / VKM B-1755 (2R,3S)-2-methylmalyl-CoA
-
r
4.2.1.148 2-methylfumaryl-CoA + H2O i.e. mesaconyl-CoA. The enzyme catalyzes a reaction in the methylaspartate cycle Haloarcula hispanica DSM 4426 (2R,3S)-2-methylmalyl-CoA
-
?
4.2.1.148 2-methylfumaryl-CoA + H2O i.e. mesaconyl-CoA, i.e. mesaconyl-C1-CoA. The equilibrium of the reaction is on the side of (2R,3S)-2-methylmalyl-CoA Haloarcula hispanica DSM 4426 (2R,3S)-2-methylmalyl-CoA
-
r
4.2.1.148 3-methylfumaryl-CoA + H2O i.e. mesaconyl-C4-CoA Haloarcula hispanica (S)-citramalyl-CoA
-
r
4.2.1.148 fumaryl-CoA + H2O
-
Haloarcula hispanica (S)-malyl-CoA
-
r
4.2.1.148 mesaconyl-C1-CoA the physiological substrate is mesaconyl-C1-CoA Haloarcula hispanica (2R,3S)-2-methyl-3-hydroxysuccinyl-C1-CoA + H2O
-
r
4.2.1.148 mesaconyl-C1-CoA hydration of mesaconyl-C1-CoA to (2R,3S)-2-methyl-3-hydroxysuccinyl-C1-CoA (erythro-beta-methylmalyl-CoA), EC 4.2.1.148. The activity for hydration of mesaconyl-C1-CoA is an order of magnitude higher for the reverse reaction of beta-methylmalyl-CoA dehydration Haloarcula hispanica (2R,3S)-2-methyl-3-hydroxysuccinyl-C1-CoA + H2O
-
r
4.2.1.148 mesaconyl-C1-CoA the physiological substrate is mesaconyl-C1-CoA Haloarcula hispanica ATCC 33960 / DSM 4426 / JCM 8911 / NBRC 102182 / NCIMB 2187 / VKM B-1755 (2R,3S)-2-methyl-3-hydroxysuccinyl-C1-CoA + H2O
-
r
4.2.1.148 mesaconyl-C1-CoA the physiological substrate is mesaconyl-C1-CoA Haloarcula hispanica DSM 4426 (2R,3S)-2-methyl-3-hydroxysuccinyl-C1-CoA + H2O
-
r
4.2.1.148 mesaconyl-C4-CoA + H2O EC 4.2.1.153 Haloarcula hispanica (2R,3S)-2-methyl-3-hydroxysuccinyl-C4-CoA + H2O
-
r
4.2.1.148 additional information recombinant mesaconyl-CoA hydratase has a 3.5fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA. The enzyme is also active with mesaconyl-C4-CoA, (S)-citramalyl-CoA and (S)-malyl-CoA, and virtually has no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. The enzyme activity is an order of magnitude higher for mesaconyl-C1-CoA/beta-methylmalyl-CoA compared to other substrates Haloarcula hispanica ?
-
?
4.2.1.148 additional information recombinant mesaconyl-CoA hydratase has a 3.5fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA. The enzyme is also active with mesaconyl-C4-CoA, (S)-citramalyl-CoA and (S)-malyl-CoA, and virtually has no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. The enzyme activity is an order of magnitude higher for mesaconyl-C1-CoA/beta-methylmalyl-CoA compared to other substrates Haloarcula hispanica ATCC 33960 / DSM 4426 / JCM 8911 / NBRC 102182 / NCIMB 2187 / VKM B-1755 ?
-
?
4.2.1.148 additional information recombinant mesaconyl-CoA hydratase has a 3.5fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA. The enzyme is also active with mesaconyl-C4-CoA, (S)-citramalyl-CoA and (S)-malyl-CoA, and virtually has no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. The enzyme activity is an order of magnitude higher for mesaconyl-C1-CoA/beta-methylmalyl-CoA compared to other substrates Haloarcula hispanica DSM 4426 ?
-
?

Subunits

EC Number Subunits Comment Organism
2.8.3.26 homodimer 2 * 48000, SDS-PAGE Haloarcula hispanica
4.2.1.148 dimer 2 * 43000, recombinant His-tagged enzyme, SDS-PAGE, 2 * 41000, about, sequence calculation Haloarcula hispanica
4.2.1.148 homodimer 2 * 43000, SDS-PAGE Haloarcula hispanica
4.2.1.148 More haloarchaeal Mch consists of two (R)-specific enoyl-CoA hydratase domains Haloarcula hispanica

Synonyms

EC Number Synonyms Comment Organism
2.8.3.26 MCT
-
Haloarcula hispanica
2.8.3.26 mesaconate:succinyl-CoA CoA transferase
-
Haloarcula hispanica
4.2.1.148 Hah_1340
-
Haloarcula hispanica
4.2.1.148 Mch
-
Haloarcula hispanica
4.2.1.148 mesaconyl-CoA hydratase
-
Haloarcula hispanica
4.2.1.148 More cf. EC 4.2.1.153 Haloarcula hispanica

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.8.3.26 37
-
assay at Haloarcula hispanica
4.2.1.148 37
-
assay at Haloarcula hispanica

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.8.3.26 12.2
-
succinyl-CoA 37°C, pH 7.8 Haloarcula hispanica
2.8.3.26 12.2
-
mesaconate 37°C, pH 7.8 Haloarcula hispanica

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.8.3.26 7.8
-
assay at Haloarcula hispanica
4.2.1.148 7.8
-
assay at Haloarcula hispanica

pI Value

EC Number Organism Comment pI Value Maximum pI Value
2.8.3.26 Haloarcula hispanica calculated from sequence
-
4.3
4.2.1.148 Haloarcula hispanica calculated from sequence
-
4.7

General Information

EC Number General Information Comment Organism
2.8.3.26 evolution the mct gene is mainly transmitted vertically among haloarchaea Haloarcula hispanica
2.8.3.26 metabolism the enzyme catalyzes a reaction in the methylaspartate cycle Haloarcula hispanica
4.2.1.148 evolution the mch gene is mainly transmitted vertically among haloarchaea Haloarcula hispanica
4.2.1.148 evolution the methylaspartate cycle is discovered in Haloarcula spp. and operates in about 40% of sequenced haloarchaea. Mesaconate CoA-transferase and mesaconyl-CoA hydratase are the characteristic enzymes of this cycle. Phylogenetic analysis Haloarcula hispanica
4.2.1.148 metabolism mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to beta-methylmalyl-CoA in the methylaspartate cycle as part of the anaplerotic pathway for the conversion of acetyl-CoA into cellular building blocks. The cleavage of beta-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another acetyl-CoA molecule give rise to two C4-dicarboxylic acids, thus regenerating the initial acetyl-CoA acceptor and forming malate, its final product. The methylaspartate cycle, overview Haloarcula hispanica
4.2.1.148 metabolism the enzyme catalyzes a reaction in the methylaspartate cycle Haloarcula hispanica

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.8.3.26 0.12
-
Acrylate 37°C, pH 7.8 Haloarcula hispanica
2.8.3.26 0.32
-
Glutarate 37°C, pH 7.8 Haloarcula hispanica
2.8.3.26 4.6
-
mesaconate 37°C, pH 7.8, 5 mM succinyl-CoA Haloarcula hispanica
2.8.3.26 9.1
-
Methylsuccinate 37°C, pH 7.8, 5 mM succinyl-CoA Haloarcula hispanica
2.8.3.26 9.8
-
mesaconate 37°C, pH 7.8, 1 mM succinyl-CoA Haloarcula hispanica
2.8.3.26 12.4
-
Methylsuccinate 37°C, pH 7.8, 1 mM succinyl-CoA Haloarcula hispanica
2.8.3.26 17.8
-
succinyl-CoA 37°C, pH 7.8 Haloarcula hispanica
4.2.1.148 0.2
-
(S)-citramalyl-CoA 37°C, pH 7.8 Haloarcula hispanica
4.2.1.148 2
-
3-methylfumaryl-CoA 37°C, pH 7.8 Haloarcula hispanica
4.2.1.148 3.8
-
(S)-malyl-CoA 37°C, pH 7.8 Haloarcula hispanica
4.2.1.148 6.9
-
2-methylfumaryl-CoA 37°C, pH 7.8 Haloarcula hispanica
4.2.1.148 254
-
(2R,3S)-2-methylmalyl-CoA 37°C, pH 7.8 Haloarcula hispanica