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Literature summary for 4.2.1.36 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
Search for more literature for 4.2.1.36

Application

Application Comment Organism
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)

Cloned (Comment) Organism
expressioon of wild-type and mutant small and large subunit proteins MJ1271 and MJ1003 in Escherichia coli strain BL21(DE3) Methanocaldococcus jannaschii

Crystallization (Commentary)

Crystallization (Comment) Organism
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

Protein Variants

Protein Variants Comment Organism
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
R26K site-directed mutagenesis of small-subunit HACN protein MJ1271, the mutant variant forms a relatively efficient IPMI enzyme Methanocaldococcus jannaschii
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
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
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

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.022
-
 (Z)-but-1-ene-1,2,4-tricarboxylate wild-type HACNMj, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.03
-
 (Z)-pent-1-ene-1,2,5-tricarboxylate wild-type HACNMj, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.036
-
 (Z)-hex-1-ene-1,2,6-tricarboxylate wild-type HACNMj, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.135
-
 (Z)-but-1-ene-1,2,4-tricarboxylate mutant R26K, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.22
-
 (Z)-but-1-ene-1,2,4-tricarboxylate mutant R26V, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.22
-
 (Z)-but-1-ene-1,2,4-tricarboxylate mutant T27A, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.269
-
 (Z)-pent-1-ene-1,2,5-tricarboxylate mutant T27A, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.3
-
 (Z)-but-1-ene-1,2,4-tricarboxylate pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.46
-
 (Z)-but-1-ene-1,2,4-tricarboxylate mutant R26V/T27Y, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.64
-
 (Z)-hex-1-ene-1,2,6-tricarboxylate mutant R26V/T27Y, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.65
-
 (Z)-hex-1-ene-1,2,6-tricarboxylate mutant T27A, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.66
-
 (Z)-hex-1-ene-1,2,6-tricarboxylate mutant R26V, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.87
-
 (Z)-pent-1-ene-1,2,5-tricarboxylate mutant R26V, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
1.6
-
 (Z)-pent-1-ene-1,2,5-tricarboxylate mutant R26V/T27Y, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii

Metals/Ions

Metals/Ions Comment Organism Structure
Zn2+ Asp13 and Cys63 side chains from each subunit coordinating Zn2+ ions in small-subunit HACN protein MJ1271 Methanocaldococcus jannaschii

Molecular Weight [Da]

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

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate Methanocaldococcus jannaschii
-
 (Z)-but-1-ene-1,2,4-tricarboxylate + H2O
-
 r

Organism

Organism UniProt Comment Textmining
Methanocaldococcus jannaschii
-
-
-

Purification (Commentary)

Purification (Comment) Organism
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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
-
 Methanocaldococcus jannaschii (Z)-but-1-ene-1,2,4-tricarboxylate + H2O
-
 r
(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
(Z)-but-1-ene-1,2,4-tricarboxylic acid
-
 Methanocaldococcus jannaschii ?
-
 ?
(Z)-hex-1-ene-1,2,6-tricarboxylate
-
 Methanocaldococcus jannaschii ?
-
 ?
(Z)-pent-1-ene-1,2,5-tricarboxylate
-
 Methanocaldococcus jannaschii ?
-
 ?
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 ?
-
 ?

Subunits

Subunits Comment Organism
dimer small-subunit HACN protein MJ1271 Methanocaldococcus jannaschii
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

Synonyms Comment Organism
HACN
-
 Methanocaldococcus jannaschii
HACNMj
-
 Methanocaldococcus jannaschii
Homoaconitase
-
 Methanocaldococcus jannaschii
MJ1271
-
 Methanocaldococcus jannaschii

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.48
-
 (Z)-but-1-ene-1,2,4-tricarboxylate mutant R26V, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.66
-
 (Z)-pent-1-ene-1,2,5-tricarboxylate wild-type HACNMj, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
0.75
-
 (Z)-but-1-ene-1,2,4-tricarboxylate wild-type HACNMj, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
1.43
-
 (Z)-but-1-ene-1,2,4-tricarboxylate mutant R26K, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
1.7
-
 (Z)-but-1-ene-1,2,4-tricarboxylate mutant R26V/T27Y, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
1.9
-
 (Z)-hex-1-ene-1,2,6-tricarboxylate mutant R26V/T27Y, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
2.2
-
 (Z)-pent-1-ene-1,2,5-tricarboxylate mutant T27A, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
2.5
-
 (Z)-but-1-ene-1,2,4-tricarboxylate mutant T27A, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
2.5
-
 (Z)-hex-1-ene-1,2,6-tricarboxylate wild-type HACNMj, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
2.8
-
 (Z)-hex-1-ene-1,2,6-tricarboxylate mutant R26V, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
4.1
-
 (Z)-hex-1-ene-1,2,6-tricarboxylate mutant T27A, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
5.8
-
 (Z)-pent-1-ene-1,2,5-tricarboxylate mutant R26V, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii
6.6
-
 (Z)-pent-1-ene-1,2,5-tricarboxylate mutant R26V/T27Y, pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii

pH Optimum

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

General Information

General Information Comment Organism
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
additional information the enzymes' stereospecific hydrolyase activity make it an attractive catalyst to produce diastereomers from unsaturated precursors Methanocaldococcus jannaschii
physiological function homoaconitase proteins catalyze the isomerization of tricarboxylates with variable chain length gamma-carboxylate groups Methanocaldococcus jannaschii

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.5
-
 (Z)-but-1-ene-1,2,4-tricarboxylate pH 7.0, temperature not specified in the publication Methanocaldococcus jannaschii