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Information on EC 4.2.1.103 - cyclohexyl-isocyanide hydratase

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EC Tree
     4 Lyases
         4.2 Carbon-oxygen lyases
             4.2.1 Hydro-lyases
                4.2.1.103 cyclohexyl-isocyanide hydratase
IUBMB Comments
The enzyme from Pseudomonas putida strain N19-2 can also catalyse the hydration of other isonitriles to the corresponding N-substituted formamides. The enzyme has no metal requirements.
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UNIPROT: Q4K977
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The expected taxonomic range for this enzyme is: Bacteria, Archaea
Synonyms
isonitrile hydratase, isocyanide hydratase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
isocyanide hydratase
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isonitrile hydratase
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cyclohexyl isocyanide hydratase
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-
-
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isocyanide hydratase
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-
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isonitrile hydratase
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-
-
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N-cyclohexylformamide hydro-lyase
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-
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additional information
the enzyme belongs to the DJ-1 superfamily
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
addition
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-
-
-
SYSTEMATIC NAME
IUBMB Comments
N-cyclohexylformamide hydro-lyase (cyclohexyl-isocyanide-forming)
The enzyme from Pseudomonas putida strain N19-2 can also catalyse the hydration of other isonitriles to the corresponding N-substituted formamides. The enzyme has no metal requirements.
CAS REGISTRY NUMBER
COMMENTARY hide
358974-06-8
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-naphthyl isocyanide + H2O
N-(2-naphthyl)formamide
show the reaction diagram
active site structure, C101 is the catalytic residue, structure-function relationship, overview
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-
?
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0203 - 0.0522
2-naphthyl isocyanide
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.092 - 0.234
2-naphthyl isocyanide
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.15 - 7.62
2-naphthyl isocyanide
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
Q4K977_PSEF5
Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5)
228
0
23876
TrEMBL
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24000
2 * 24158, N-terminally GSH-tagged enzyme, sequence calculation, 2 * 24000, SDS-PAGE
24158
2 * 24158, N-terminally GSH-tagged enzyme, sequence calculation, 2 * 24000, SDS-PAGE
43700
recombinant ICH, sedimentation equilibrium ultracentrifugation
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
2 * 24158, N-terminally GSH-tagged enzyme, sequence calculation, 2 * 24000, SDS-PAGE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C101A
site-directed mutagenesis, inactive mutant
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
D17E
site-directed mutagenesis, the mutant is almost inactive
D17N
site-directed mutagenesis, the mutant is almost inactive
D17V
site-directed mutagenesis, the mutant is almost inactive
T102S
site-directed mutagenesis, the mutant shows about 2fold increased activity compared to the wild-type enzyme
T102V
site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme, the mutation results in a substrate-inhibited enzyme
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of N-terminally His-tagged wild-type and mutant ICHs in Escherichia coli strain BL21(DE3)
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
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
J. Biol. Chem.
285
29651-29661
2010
Pseudomonas fluorescens (Q4K977), Pseudomonas fluorescens, Pseudomonas fluorescens Pf-5 (Q4K977)
Manually annotated by BRENDA team