EC Number | Application | Comment | Organism |
---|---|---|---|
4.2.1.36 | synthesis | 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 | Methanocaldococcus jannaschii |
EC Number | Cloned (Comment) | Organism |
---|---|---|
4.2.1.36 | expressioon of wild-type and mutant small and large subunit proteins MJ1271 and MJ1003 in Escherichia coli strain BL21(DE3) | Methanocaldococcus jannaschii |
4.2.1.114 | small-subunit HACN protein MJ1271 is expressed in Escherichia coli BL21 CodonPlus (DE3)-RIL cells | Methanocaldococcus jannaschii |
EC Number | Crystallization (Comment) | Organism |
---|---|---|
4.2.1.36 | 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 | Methanocaldococcus jannaschii |
4.2.1.114 | 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 | Methanocaldococcus jannaschii |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
4.2.1.36 | additional information | site-directed mutagenesis of small-subunit HACN protein MJ1271 produces loop-region variant proteins that are reconstituted with wild-type MJ1003 large-subunit protein. The heteromers form promiscuous hydro-lyases with reduced activity but broader substrate specificity, overview | Methanocaldococcus jannaschii |
4.2.1.36 | R26K | site-directed mutagenesis of small-subunit HACN protein MJ1271, the mutant variant forms a relatively efficient IPMI enzyme | Methanocaldococcus jannaschii |
4.2.1.36 | R26V | 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 | Methanocaldococcus jannaschii |
4.2.1.36 | R26V/T27Y | 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 | Methanocaldococcus jannaschii |
4.2.1.36 | T27A | 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 | Methanocaldococcus jannaschii |
4.2.1.114 | R26K | the variant forms an relatively efficient isopropylmalate isomerase enzyme | Methanocaldococcus jannaschii |
4.2.1.114 | R26V | the variant forms an relatively efficient isopropylmalate isomerase enzyme | Methanocaldococcus jannaschii |
4.2.1.114 | 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 | Methanocaldococcus jannaschii |
4.2.1.114 | T27A t | the variant has uniformly lower specificity constants for both isopropylmalate isomerase and methanogen homoaconitase substrates | Methanocaldococcus jannaschii |
EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
4.2.1.36 | 0.022 | - |
(Z)-but-1-ene-1,2,4-tricarboxylate | wild-type HACNMj, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.03 | - |
(Z)-pent-1-ene-1,2,5-tricarboxylate | wild-type HACNMj, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.036 | - |
(Z)-hex-1-ene-1,2,6-tricarboxylate | wild-type HACNMj, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.135 | - |
(Z)-but-1-ene-1,2,4-tricarboxylate | mutant R26K, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.22 | - |
(Z)-but-1-ene-1,2,4-tricarboxylate | mutant R26V, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.22 | - |
(Z)-but-1-ene-1,2,4-tricarboxylate | mutant T27A, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.269 | - |
(Z)-pent-1-ene-1,2,5-tricarboxylate | mutant T27A, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.3 | - |
(Z)-but-1-ene-1,2,4-tricarboxylate | pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.46 | - |
(Z)-but-1-ene-1,2,4-tricarboxylate | mutant R26V/T27Y, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.64 | - |
(Z)-hex-1-ene-1,2,6-tricarboxylate | mutant R26V/T27Y, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.65 | - |
(Z)-hex-1-ene-1,2,6-tricarboxylate | mutant T27A, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.66 | - |
(Z)-hex-1-ene-1,2,6-tricarboxylate | mutant R26V, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.87 | - |
(Z)-pent-1-ene-1,2,5-tricarboxylate | mutant R26V, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 1.6 | - |
(Z)-pent-1-ene-1,2,5-tricarboxylate | mutant R26V/T27Y, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.022 | - |
but-1-ene-1,2,4-tricarboxylic acid | wild type enzyme | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.03 | - |
(1E)-pent-1-ene-1,2,5-tricarboxylic acid | wild type enzyme | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.036 | - |
(1E)-hex-1-ene-1,2,6-tricarboxylic acid | wild type enzyme | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.135 | - |
but-1-ene-1,2,4-tricarboxylic acid | mutant enzyme R26K | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.22 | - |
but-1-ene-1,2,4-tricarboxylic acid | mutant enzyme R26V | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.22 | - |
but-1-ene-1,2,4-tricarboxylic acid | mutant enzyme T27A | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.269 | - |
(1E)-pent-1-ene-1,2,5-tricarboxylic acid | mutant enzyme T27A | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.46 | - |
but-1-ene-1,2,4-tricarboxylic acid | mutant enzyme R26V/T27Y | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.64 | - |
(1E)-hex-1-ene-1,2,6-tricarboxylic acid | mutant enzyme R26V/T27Y | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.65 | - |
(1E)-hex-1-ene-1,2,6-tricarboxylic acid | mutant enzyme T27A | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.66 | - |
(1E)-hex-1-ene-1,2,6-tricarboxylic acid | mutant enzyme R26V | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.87 | - |
(1E)-pent-1-ene-1,2,5-tricarboxylic acid | mutant enzyme R26V | Methanocaldococcus jannaschii | |
4.2.1.114 | 1.6 | - |
(1E)-pent-1-ene-1,2,5-tricarboxylic acid | mutant enzyme R26V/T27Y | Methanocaldococcus jannaschii |
EC Number | Metals/Ions | Comment | Organism | Structure |
---|---|---|---|---|
4.2.1.36 | Zn2+ | Asp13 and Cys63 side chains from each subunit coordinating Zn2+ ions in small-subunit HACN protein MJ1271 | Methanocaldococcus jannaschii |
EC Number | Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|---|
4.2.1.36 | 143000 | - |
recombinant wild-type HACN, gel filtration | Methanocaldococcus jannaschii |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
4.2.1.36 | (1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate | Methanocaldococcus jannaschii | - |
(Z)-but-1-ene-1,2,4-tricarboxylate + H2O | - |
r |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
4.2.1.36 | Methanocaldococcus jannaschii | - |
- |
- |
4.2.1.114 | Methanocaldococcus jannaschii | - |
- |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
4.2.1.36 | 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 | Methanocaldococcus jannaschii |
4.2.1.114 | Toyopearl SuperQ-650 M column chromatography, Resource S column chromatography, and Superdex 200 gel filtration | Methanocaldococcus jannaschii |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
4.2.1.36 | (1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate | - |
Methanocaldococcus jannaschii | (Z)-but-1-ene-1,2,4-tricarboxylate + H2O | - |
r | |
4.2.1.36 | (Z)-but-1-ene-1,2,4-tricarboxylate + H2O | i.e. cis-homoaconitate, three different stereoisomeric substrate types, cis-homo1-aconitate, cis-homo2-aconitate, and cis-homo3-aconitate, in the reaction, overview | Methanocaldococcus jannaschii | (1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate | 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 | r | |
4.2.1.36 | (Z)-but-1-ene-1,2,4-tricarboxylic acid | - |
Methanocaldococcus jannaschii | ? | - |
? | |
4.2.1.36 | (Z)-hex-1-ene-1,2,6-tricarboxylate | - |
Methanocaldococcus jannaschii | ? | - |
? | |
4.2.1.36 | (Z)-pent-1-ene-1,2,5-tricarboxylate | - |
Methanocaldococcus jannaschii | ? | - |
? | |
4.2.1.36 | additional information | HACNMj is specific for cis-unsaturated tricarboxylates, while isopropylmalate isomerase, IPMIMj, recognizes cis-unsaturated dicarboxylates, substrate specificity determinants of homologous IPMI and HACN proteins from Methanocaldococcus jannaschii from a structural model show characteristic residues in a flexible loop region between R2 and R3 that distinguish HACN from IPMI and aconitase proteins, overview | Methanocaldococcus jannaschii | ? | - |
? | |
4.2.1.114 | (1E)-hex-1-ene-1,2,6-tricarboxylic acid + H2O | - |
Methanocaldococcus jannaschii | (2R)-hexane-1,2,6-tricarboxyclic acid | - |
? | |
4.2.1.114 | (1E)-pent-1-ene-1,2,5-tricarboxylic acid + H2O | - |
Methanocaldococcus jannaschii | (2R)-pentane-1,2,5-tricarboxylic acid | - |
? | |
4.2.1.114 | (2R)-butane-1,2,4-tricarboxylic acid | - |
Methanocaldococcus jannaschii | but-1-ene-1,2,4-tricarboxylic acid + H2O | - |
? | |
4.2.1.114 | but-1-ene-1,2,4-tricarboxylic acid + H2O | - |
Methanocaldococcus jannaschii | (2R)-butane-1,2,4-tricarboxylic acid | - |
? | |
4.2.1.114 | dihomocitrate | - |
Methanocaldococcus jannaschii | cis-(homo)2aconitate + H2O | - |
? | |
4.2.1.114 | additional information | HACN has no detectable activity with citraconate or isopropylmalate | Methanocaldococcus jannaschii | ? | - |
? | |
4.2.1.114 | trihomocitrate | - |
Methanocaldococcus jannaschii | cis-(homo)3aconitate + H2O | - |
? |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
4.2.1.36 | dimer | small-subunit HACN protein MJ1271 | Methanocaldococcus jannaschii |
4.2.1.36 | More | 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 | Methanocaldococcus jannaschii |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
4.2.1.36 | HACN | - |
Methanocaldococcus jannaschii |
4.2.1.36 | HACNMj | - |
Methanocaldococcus jannaschii |
4.2.1.36 | Homoaconitase | - |
Methanocaldococcus jannaschii |
4.2.1.36 | MJ1271 | - |
Methanocaldococcus jannaschii |
4.2.1.114 | HACN | - |
Methanocaldococcus jannaschii |
4.2.1.114 | MJ1003 | large subunit protein of HACN | Methanocaldococcus jannaschii |
4.2.1.114 | MJ1271 | small subunit protein of HACN | Methanocaldococcus jannaschii |
EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
4.2.1.36 | 0.48 | - |
(Z)-but-1-ene-1,2,4-tricarboxylate | mutant R26V, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.66 | - |
(Z)-pent-1-ene-1,2,5-tricarboxylate | wild-type HACNMj, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 0.75 | - |
(Z)-but-1-ene-1,2,4-tricarboxylate | wild-type HACNMj, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 1.43 | - |
(Z)-but-1-ene-1,2,4-tricarboxylate | mutant R26K, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 1.7 | - |
(Z)-but-1-ene-1,2,4-tricarboxylate | mutant R26V/T27Y, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 1.9 | - |
(Z)-hex-1-ene-1,2,6-tricarboxylate | mutant R26V/T27Y, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 2.2 | - |
(Z)-pent-1-ene-1,2,5-tricarboxylate | mutant T27A, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 2.5 | - |
(Z)-but-1-ene-1,2,4-tricarboxylate | mutant T27A, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 2.5 | - |
(Z)-hex-1-ene-1,2,6-tricarboxylate | wild-type HACNMj, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 2.8 | - |
(Z)-hex-1-ene-1,2,6-tricarboxylate | mutant R26V, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 4.1 | - |
(Z)-hex-1-ene-1,2,6-tricarboxylate | mutant T27A, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 5.8 | - |
(Z)-pent-1-ene-1,2,5-tricarboxylate | mutant R26V, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.36 | 6.6 | - |
(Z)-pent-1-ene-1,2,5-tricarboxylate | mutant R26V/T27Y, pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.48 | - |
but-1-ene-1,2,4-tricarboxylic acid | mutant enzyme R26V | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.66 | - |
(1E)-pent-1-ene-1,2,5-tricarboxylic acid | wild type enzyme | Methanocaldococcus jannaschii | |
4.2.1.114 | 0.75 | - |
but-1-ene-1,2,4-tricarboxylic acid | wild type enzyme | Methanocaldococcus jannaschii | |
4.2.1.114 | 1.43 | - |
but-1-ene-1,2,4-tricarboxylic acid | mutant enzyme R26K | Methanocaldococcus jannaschii | |
4.2.1.114 | 1.7 | - |
but-1-ene-1,2,4-tricarboxylic acid | mutant enzyme R26V/T27Y | Methanocaldococcus jannaschii | |
4.2.1.114 | 1.9 | - |
(1E)-hex-1-ene-1,2,6-tricarboxylic acid | mutant enzyme R26V/T27Y | Methanocaldococcus jannaschii | |
4.2.1.114 | 2.2 | - |
(1E)-pent-1-ene-1,2,5-tricarboxylic acid | mutant enzyme T27A | Methanocaldococcus jannaschii | |
4.2.1.114 | 2.5 | - |
(1E)-hex-1-ene-1,2,6-tricarboxylic acid | wild type enzyme | Methanocaldococcus jannaschii | |
4.2.1.114 | 2.5 | - |
but-1-ene-1,2,4-tricarboxylic acid | mutant enzyme T27A | Methanocaldococcus jannaschii | |
4.2.1.114 | 2.8 | - |
(1E)-hex-1-ene-1,2,6-tricarboxylic acid | mutant enzyme R26V | Methanocaldococcus jannaschii | |
4.2.1.114 | 4.1 | - |
(1E)-hex-1-ene-1,2,6-tricarboxylic acid | mutant enzyme T27A | Methanocaldococcus jannaschii | |
4.2.1.114 | 5.8 | - |
(1E)-pent-1-ene-1,2,5-tricarboxylic acid | mutant enzyme R26V | Methanocaldococcus jannaschii | |
4.2.1.114 | 6.6 | - |
(1E)-pent-1-ene-1,2,5-tricarboxylic acid | mutant enzyme R26V/T27Y | Methanocaldococcus jannaschii |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
4.2.1.36 | 7 | - |
assay at | Methanocaldococcus jannaschii |
EC Number | General Information | Comment | Organism |
---|---|---|---|
4.2.1.36 | evolution | 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 | Methanocaldococcus jannaschii |
4.2.1.36 | additional information | the enzymes' stereospecific hydrolyase activity make it an attractive catalyst to produce diastereomers from unsaturated precursors | Methanocaldococcus jannaschii |
4.2.1.36 | physiological function | homoaconitase proteins catalyze the isomerization of tricarboxylates with variable chain length gamma-carboxylate groups | Methanocaldococcus jannaschii |
EC Number | kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
4.2.1.36 | 2.5 | - |
(Z)-but-1-ene-1,2,4-tricarboxylate | pH 7.0, temperature not specified in the publication | Methanocaldococcus jannaschii |