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Literature summary extracted from

  • 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 (2010), Biochemistry, 49, 2687-2696.
    View publication on PubMed

Application

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

Cloned(Commentary)

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

Crystallization (Commentary)

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

Protein Variants

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

KM Value [mM]

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

Metals/Ions

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

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
4.2.1.36 143000
-
recombinant wild-type HACN, gel filtration Methanocaldococcus jannaschii

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
4.2.1.36 Methanocaldococcus jannaschii
-
-
-
4.2.1.114 Methanocaldococcus jannaschii
-
-
-

Purification (Commentary)

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

Substrates and Products (Substrate)

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
-
?

Subunits

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

Synonyms

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

Turnover Number [1/s]

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

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.1.36 7
-
assay at Methanocaldococcus jannaschii

General Information

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

kcat/KM [mM/s]

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