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Literature summary extracted from

  • Lakshminarasimhan, M.; Madzelan, P.; Nan, R.; Milkovic, N.M.; Wilson, M.A.
    Evolution of new enzymatic function by structural modulation of cysteine reactivity in Pseudomonas fluorescens isocyanide hydratase (2010), J. Biol. Chem., 285, 29651-29661.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.1.103 expression of N-terminally His-tagged wild-type and mutant ICHs in Escherichia coli strain BL21(DE3) Pseudomonas fluorescens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.2.1.103 purified recombinant wild-type and several site-directed mutants, hanging drop vapor diffusion method, for wild-type, C101S, and C101A enzymes: 0.002 ml of 20 mg/ml protein solution is mixed with 0.002 ml of reservoir solution containing 24-26% PEG 3350, 200-250 mM magnesium chloride, 100 mM CHES, pH 9.3, or 100 mM Tris-HCl, pH 8.6, and equilibration against 0.5 ml of reservoir solution, 1-3 days, for D17E enzyme: 0.002 ml of 24 mg/ml protein is mixed with 0.002 ml of reservoir solution containing 12% PEG 4000, 240 mM ammonium acetate, 100 mM sodium acetate, pH 4.6, 2-3 days at room temperature, for T102S enzyme: 0.002 ml of 17 mg/ml protein solution is mixed with 0.002 ml of reservoir solution containing 19-22% PEG 4000, 140-160 mM sodium citrate, pH 5.6, 2-3 days at room temperature, X-ray diffraction structure determination and analysis at 1.0-1.9 A resolution Pseudomonas fluorescens

Protein Variants

EC Number Protein Variants Comment Organism
4.2.1.103 C101A site-directed mutagenesis, inactive mutant Pseudomonas fluorescens
4.2.1.103 C101S site-directed mutagenesis, inactive mutant, the mutant shows a stronger electron density for a water molecule between Asp17 and residue 101 compared to the wild-type enzyme Pseudomonas fluorescens
4.2.1.103 D17E site-directed mutagenesis, the mutant is almost inactive Pseudomonas fluorescens
4.2.1.103 D17N site-directed mutagenesis, the mutant is almost inactive Pseudomonas fluorescens
4.2.1.103 D17V site-directed mutagenesis, the mutant is almost inactive Pseudomonas fluorescens
4.2.1.103 T102S site-directed mutagenesis, the mutant shows about 2fold increased activity compared to the wild-type enzyme Pseudomonas fluorescens
4.2.1.103 T102V site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme, the mutation results in a substrate-inhibited enzyme Pseudomonas fluorescens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.2.1.103 0.0203
-
 2-naphthyl isocyanide pH 7.4, 25°C, recombinant mutant T102V Pseudomonas fluorescens
4.2.1.103 0.0207
-
 2-naphthyl isocyanide pH 7.4, 25°C, recombinant mutant T102S Pseudomonas fluorescens
4.2.1.103 0.0522
-
 2-naphthyl isocyanide pH 7.4, 25°C, recombinant wild-type ICH Pseudomonas fluorescens

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
4.2.1.103 24000
-
 2 * 24158, N-terminally GSH-tagged enzyme, sequence calculation, 2 * 24000, SDS-PAGE Pseudomonas fluorescens
4.2.1.103 24158
-
 2 * 24158, N-terminally GSH-tagged enzyme, sequence calculation, 2 * 24000, SDS-PAGE Pseudomonas fluorescens
4.2.1.103 43700
-
 recombinant ICH, sedimentation equilibrium ultracentrifugation Pseudomonas fluorescens

Organism

EC Number Organism UniProt Comment Textmining
4.2.1.103 Pseudomonas fluorescens Q4K977
-
-
4.2.1.103 Pseudomonas fluorescens Pf-5 Q4K977
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.1.103 recombinant N-terminally His-tagged wild-type and mutant ICHs from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, cleavage of the His-tag through thrombin, and benzamidine affinity chromatography. The final recombinant ICH protein has three vector-derived amino acids at the N terminus, GSH Pseudomonas fluorescens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.1.103 2-naphthyl isocyanide + H2O active site structure, C101 is the catalytic residue, structure-function relationship, overview Pseudomonas fluorescens N-(2-naphthyl)formamide
-
 ?
4.2.1.103 2-naphthyl isocyanide + H2O active site structure, C101 is the catalytic residue, structure-function relationship, overview Pseudomonas fluorescens Pf-5 N-(2-naphthyl)formamide
-
 ?

Subunits

EC Number Subunits Comment Organism
4.2.1.103 homodimer 2 * 24158, N-terminally GSH-tagged enzyme, sequence calculation, 2 * 24000, SDS-PAGE Pseudomonas fluorescens

Synonyms

EC Number Synonyms Comment Organism
4.2.1.103 ICH
-
 Pseudomonas fluorescens
4.2.1.103 isocyanide hydratase
-
 Pseudomonas fluorescens
4.2.1.103 isonitrile hydratase
-
 Pseudomonas fluorescens
4.2.1.103 More the enzyme belongs to the DJ-1 superfamily Pseudomonas fluorescens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.1.103 25
-
 assay at Pseudomonas fluorescens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.2.1.103 0.092
-
 2-naphthyl isocyanide pH 7.4, 25°C, recombinant mutant T102V Pseudomonas fluorescens
4.2.1.103 0.216
-
 2-naphthyl isocyanide pH 7.4, 25°C, recombinant wild-type ICH Pseudomonas fluorescens
4.2.1.103 0.234
-
 2-naphthyl isocyanide pH 7.4, 25°C, recombinant mutant T102S Pseudomonas fluorescens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.1.103 7.4
-
 assay at Pseudomonas fluorescens

General Information

EC Number General Information Comment Organism
4.2.1.103 physiological function isocyanide hydratase is an enzyme of the DJ-1 superfamily that hydrates isocyanides to yield the corresponding N-formamide, structural basis for catalysis, overview. ICH contains a highly conserved Cys101 that is required for catalysis and interacts with Asp17, Thr102, and an ordered water molecule in the active site. Asp17 activates the ordered water molecule to hydrate organic isocyanides. The thiolate of Cys101 is proposed to be a critical nucleophile that initiates the hydration of isocyanides Pseudomonas fluorescens

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
4.2.1.103 4.15
-
 2-naphthyl isocyanide pH 7.4, 25°C, recombinant wild-type ICH Pseudomonas fluorescens
4.2.1.103 4.53
-
 2-naphthyl isocyanide pH 7.4, 25°C, recombinant mutant T102V Pseudomonas fluorescens
4.2.1.103 7.62
-
 2-naphthyl isocyanide pH 7.4, 25°C, recombinant mutant T102S Pseudomonas fluorescens