Literature summary for 4.2.1.103 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.

Search for more literature for 4.2.1.103

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
C101A	site-directed mutagenesis, inactive mutant	Pseudomonas fluorescens
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
D17E	site-directed mutagenesis, the mutant is almost inactive	Pseudomonas fluorescens
D17N	site-directed mutagenesis, the mutant is almost inactive	Pseudomonas fluorescens
D17V	site-directed mutagenesis, the mutant is almost inactive	Pseudomonas fluorescens
T102S	site-directed mutagenesis, the mutant shows about 2fold increased activity compared to the wild-type enzyme	Pseudomonas fluorescens
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]

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

Molecular Weight [Da]

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

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas fluorescens	Q4K977	-	-
Pseudomonas fluorescens Pf-5	Q4K977	-	-

Purification (Commentary)

Purification (Comment)	Organism
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)

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

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

Synonyms

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

General Information

General Information	Comment	Organism
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]

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