Cloned (Comment) | Organism |
---|---|
gene Hah_1340, phylogenetic analysis, recombinant expression of soluble His-tagged enzyme in Haloferax volcanii strain H1209 | Haloarcula hispanica |
Haloferax volcanii strain H1209 is used for the heterologous expression of the Haloarcula hispanica proteins | Haloarcula hispanica |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
CaCl2 | 18% inhibition at 5 mM; 5 mM, 18% inhibition | Haloarcula hispanica | |
CoCl2 | 40% inhibition at 5 mM; 5 mM, 34% inhibition | Haloarcula hispanica | |
MgCl2 | 11% inhibition at 5 mM; 5 mM, 11% inhibition | Haloarcula hispanica | |
MnCl2 | 22% inhibition at 5 mM; 5 mM, 22% inhibition | Haloarcula hispanica | |
additional information | no inhibition by EDTA; no inhibition by EDTA at 2 mM | Haloarcula hispanica | |
NiCl2 | 34% inhibition at 5 mM; 5 mM, 40% inhibition | Haloarcula hispanica |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.18 | - |
3-methylfumaryl-CoA | 37°C, pH 7.8 | Haloarcula hispanica | |
0.32 | - |
(S)-malyl-CoA | 37°C, pH 7.8 | Haloarcula hispanica | |
0.35 | - |
(2R,3S)-2-methylmalyl-CoA | 37°C, pH 7.8 | Haloarcula hispanica | |
1 | - |
2-methylfumaryl-CoA | 37°C, pH 7.8 | Haloarcula hispanica | |
2.6 | - |
(S)-citramalyl-CoA | 37°C, pH 7.8 | Haloarcula hispanica |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
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 | |
additional information | no requirement for divalent cations | Haloarcula hispanica |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
86000 | - |
gel filtration | Haloarcula hispanica |
86000 | - |
recombinant His-tagged enzyme, gel filtration | Haloarcula hispanica |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-methylfumaryl-CoA + H2O | Haloarcula hispanica | i.e. mesaconyl-CoA. The enzyme catalyzes a reaction in the methylaspartate cycle | (2R,3S)-2-methylmalyl-CoA | - |
? | |
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 | - |
? | |
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 | - |
? | |
mesaconyl-C1-CoA | Haloarcula hispanica | the physiological substrate is mesaconyl-C1-CoA | (2R,3S)-2-methyl-3-hydroxysuccinyl-C1-CoA + H2O | - |
r | |
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 | |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Haloarcula hispanica | G0HQ35 | - |
- |
Haloarcula hispanica ATCC 33960 / DSM 4426 / JCM 8911 / NBRC 102182 / NCIMB 2187 / VKM B-1755 | G0HQ35 | - |
- |
Haloarcula hispanica DSM 4426 | G0HQ35 | - |
- |
Purification (Comment) | Organism |
---|---|
- |
Haloarcula hispanica |
recombinant His-tagged enzyme 427fold from Haloferax volcanii strain H1209 by nickel affinity chromatography and gel filtration | Haloarcula hispanica |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
157 | - |
37°C, pH 7.8 | Haloarcula hispanica |
157 | - |
purified recombinant His-tagged enzyme, substrate mesaconyl-C1-CoA, pH 7.8, 37°C | Haloarcula hispanica |
Storage Stability | Organism |
---|---|
-20°C, purified recombinant enzyme, 50 mM Tris/HCl, pH 8.0, 2 M KCl, 3 weeks, complete loss of activity | Haloarcula hispanica |
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(2R,3S)-2-methyl-3-hydroxysuccinyl-C4-CoA + H2O | i.e. (S)-citramalyl-CoA | Haloarcula hispanica | 3-methylfumaryl-CoA + H2O | - |
r | |
(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 | |
(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 | |
(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 | |
(S)-citramalyl-CoA | - |
Haloarcula hispanica | 3-methylfumaryl-CoA + H2O | - |
r | |
(S)-malyl-CoA | - |
Haloarcula hispanica | fumaryl-CoA + H2O | - |
r | |
2-methylfumaryl-CoA + H2O | i.e. mesaconyl-CoA. The enzyme catalyzes a reaction in the methylaspartate cycle | Haloarcula hispanica | (2R,3S)-2-methylmalyl-CoA | - |
? | |
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 | |
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 | - |
? | |
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 | |
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 | - |
? | |
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 | |
3-methylfumaryl-CoA + H2O | i.e. mesaconyl-C4-CoA | Haloarcula hispanica | (S)-citramalyl-CoA | - |
r | |
fumaryl-CoA + H2O | - |
Haloarcula hispanica | (S)-malyl-CoA | - |
r | |
mesaconyl-C1-CoA | the physiological substrate is mesaconyl-C1-CoA | Haloarcula hispanica | (2R,3S)-2-methyl-3-hydroxysuccinyl-C1-CoA + H2O | - |
r | |
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 | |
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 | |
mesaconyl-C1-CoA | the physiological substrate is mesaconyl-C1-CoA | Haloarcula hispanica DSM 4426 | (2R,3S)-2-methyl-3-hydroxysuccinyl-C1-CoA + H2O | - |
r | |
mesaconyl-C4-CoA + H2O | EC 4.2.1.153 | Haloarcula hispanica | (2R,3S)-2-methyl-3-hydroxysuccinyl-C4-CoA + H2O | - |
r | |
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 | ? | - |
? | |
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 | ? | - |
? | |
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 | Comment | Organism |
---|---|---|
dimer | 2 * 43000, recombinant His-tagged enzyme, SDS-PAGE, 2 * 41000, about, sequence calculation | Haloarcula hispanica |
homodimer | 2 * 43000, SDS-PAGE | Haloarcula hispanica |
More | haloarchaeal Mch consists of two (R)-specific enoyl-CoA hydratase domains | Haloarcula hispanica |
Synonyms | Comment | Organism |
---|---|---|
Hah_1340 | - |
Haloarcula hispanica |
Mch | - |
Haloarcula hispanica |
mesaconyl-CoA hydratase | - |
Haloarcula hispanica |
More | cf. EC 4.2.1.153 | Haloarcula hispanica |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Haloarcula hispanica |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.8 | - |
assay at | Haloarcula hispanica |
Organism | Comment | pI Value Maximum | pI Value |
---|---|---|---|
Haloarcula hispanica | calculated from sequence | - |
4.7 |
General Information | Comment | Organism |
---|---|---|
evolution | the mch gene is mainly transmitted vertically among haloarchaea | Haloarcula hispanica |
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 |
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 |
metabolism | the enzyme catalyzes a reaction in the methylaspartate cycle | Haloarcula hispanica |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.2 | - |
(S)-citramalyl-CoA | 37°C, pH 7.8 | Haloarcula hispanica | |
2 | - |
3-methylfumaryl-CoA | 37°C, pH 7.8 | Haloarcula hispanica | |
3.8 | - |
(S)-malyl-CoA | 37°C, pH 7.8 | Haloarcula hispanica | |
6.9 | - |
2-methylfumaryl-CoA | 37°C, pH 7.8 | Haloarcula hispanica | |
254 | - |
(2R,3S)-2-methylmalyl-CoA | 37°C, pH 7.8 | Haloarcula hispanica |