Information on EC 4.1.2.46 - aliphatic (R)-hydroxynitrile lyase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.1.2.46
-
RECOMMENDED NAME
GeneOntology No.
aliphatic (R)-hydroxynitrile lyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(2R)-2-hydroxy-2-methylbutanenitrile = cyanide + butan-2-one
show the reaction diagram
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
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Cyanoamino acid metabolism
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linamarin degradation
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linustatin bioactivation
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lotaustralin degradation
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neolinustatin bioactivation
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SYSTEMATIC NAME
IUBMB Comments
(2R)-2-hydroxy-2-methylbutanenitrile butan-2-one lyase (cyanide forming)
The enzyme contains Zn2+ [1]. The enzyme catalyses the stereoselective synthesis of aliphatic (R)-cyanohydrins [1]. No activity towards mandelonitrile and 4-hydroxymandelonitrile [5]. Natural substrates for the (R)-oxynitrilase from Linum usitatissimum are acetone and butan-2-one, which are the building blocks of the cyanogen glycosides in Linum, linamarin and lotaustralin, or linustatin and neolinustatin, respectively [4].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2R)-2-hydroxy-2-methylbutanenitrile
cyanide + 2-butanone
show the reaction diagram
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-
-
-
?
(R)-2-butanone-cyanhydrin
HCN + butanone
show the reaction diagram
-
-
-
-
?
2-hydroxy-2-methylpropanenitrile
cyanide + acetone
show the reaction diagram
3,3-dimethyl-2-butanone + acetone cyanohydrin
2-hydroxy-2,3,3-trimethylbutanenitrile + acetone
show the reaction diagram
-
-
-
-
?
acetyltrimethylsilane + acetone cyanohydrin
(R)-2-trimethylsilyl-2-hydroxy-ethylcyanide + acetone
show the reaction diagram
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enantioselective transcyanation. Under optimum conditions, both acetyltrimethylsilane conversion to (R)-2-trimethylsilyl-2-hydroxy-ethylcyanide and enantiomeric excess of the product are above 99%. The silicon atom in acetyltrimethylsilane has a great effect on the eaction and both the substrate conversion and the product enantiomeric excess are much higher than those in its carbon counterpart 3,3-dimethyl-2-butanone
-
?
cyanide + 2-methylcyclopentanone
?
show the reaction diagram
-
-
-
?
cyanide + 2-pentanone
(2R)-2-hydroxy-2-methylpentanenitrile
show the reaction diagram
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93% enantiomeric excess
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?
cyanide + acetone
2-hydroxy-2-methylpropanenitrile
show the reaction diagram
cyanide + acetylcyclopropane
?
show the reaction diagram
-
-
-
?
cyanide + acrolein
(2R)-2-hydroxybut-3-enenitrile
show the reaction diagram
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74% enantiomeric excess
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?
cyanide + butan-2-one
(2R)-2-hydroxy-2-methylbutanenitrile
show the reaction diagram
cyanide + butan-2-one
(2R)-butan-2-one cyanohydrin
show the reaction diagram
natural substrates for the (R)-oxynitrilase from Linum usitatissimum are acetone and butan-2-one, which are the building blocks of the cyanogen glycosides in Linum, linamarin and lotaustralin, or linustatin and neolinustatin, respectively
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-
?
cyanide + butyraldehyde
(2R)-2-hydroxypentanenitrile
show the reaction diagram
cyanide + chloroacetone
(2R)-3-chloro-2-hydroxy-2-methylpropionitrile
show the reaction diagram
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-
-
?
cyanide + crotonaldehyde
(2R)-2-hydroxy-3-pentenenitrile
show the reaction diagram
cyanide + hexan-2-one
(2R)-2-hydroxy-2-methylhexanenitrile
show the reaction diagram
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-
-
?
cyanide + hydroxyacetone
(2R)-1,2-dihydroxy-2-methyl-propane-3-nitrile
show the reaction diagram
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-
-
?
cyanide + hydroxypivaldehyde
(2R)-2,4-dihydroxy-3,3-dimethylbutanenitrile
show the reaction diagram
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73% enantiomeric excess
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?
cyanide + isobutyraldehyde
(2R)-2-hydroxy-4-methylpentanenitrile
show the reaction diagram
-
93% enantiomeric excess
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?
cyanide + methacrolein
(2R)-2-hydroxy-3-methylbut-3-enenitrile
show the reaction diagram
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98% enantiomeric excess
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?
cyanide + methyl vinyl ketone
(2R)-2-hydroxy-2-methyl-3-butenenitrile
show the reaction diagram
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-
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?
cyanide + methyl vinyl ketone
(2R)-2-hydroxy-2-methylbut-3-enenitrile
show the reaction diagram
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despite a short reaction time of 0.8 h, the conversion of methyl vinyl ketone results in a poor (38%) enantiomeric excess value. As in the same time there is almost no conversion without enzyme. This compound is one of the rare examples, where the enzyme exerts only a partial stereoselectivity for a defined substrate
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?
cyanide + pentan-2,4-dione
?
show the reaction diagram
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-
-
?
cyanide + pentan-2-one
(2R)-2-hydroxy-2-methylpentanenitrile
show the reaction diagram
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-
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?
cyanide + pinacolone
(2R)-2-hydroxy-2,3,3-trimethylbutyronitrile
show the reaction diagram
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-
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?
cyanide + pivalaldehyde
(2R)-3,3-dimethyl-2-hydroxybutyronitrile
show the reaction diagram
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?
cyanide + propionaldehyde
(2R)-2-hydroxybutyronitrile
show the reaction diagram
cyanide + pyruvic acid ethyl ester
?
show the reaction diagram
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?
HCN + 4-hydroxybutanal
2,5-dihydroxypentanenitrile
show the reaction diagram
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?
HCN + benzaldehyde
(R)-mandelonitrile
show the reaction diagram
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
2-hydroxy-2-methylpropanenitrile
cyanide + acetone
show the reaction diagram
cyanide + acetone
2-hydroxy-2-methylpropanenitrile
show the reaction diagram
P93243
natural substrates for the (R)-oxynitrilase from Linum usitatissimum are acetone and butan-2-one, which are the building blocks of the cyanogenic glycosides in Linum, linamarin and lotaustralin, or linustatin and neolinustatin, respectively
-
-
?
cyanide + butan-2-one
(2R)-butan-2-one cyanohydrin
show the reaction diagram
P93243
natural substrates for the (R)-oxynitrilase from Linum usitatissimum are acetone and butan-2-one, which are the building blocks of the cyanogen glycosides in Linum, linamarin and lotaustralin, or linustatin and neolinustatin, respectively
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-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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not a flavoprotein
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Zn2+
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LuHNL has significant homologies to members of the Zn2+-containing alcohol dehydrogenases. In particular, residues responsible for coordination of Zn2+ ions or fulfilling structural or functional tasks in Zn2+-alcohol dehydrogenases are conserved. Contains about 2-4 mol zinc per mol of recombinant enzyme. Hydroxynitrile lyase from Linum usitatissimum and Zn2+-alcohol dehydrogenases have similar structural requirements with respect to maintaining a catalytically active structure. Residues essentially involved in catalysis of Zn2+-ADHs are also of functional importance in hydroxynitrile lyase from Linum usitatissimum
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
benzaldehyde
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leads to a complete and irreversible deactivation of the enzyme within 2 h incubation
diisopropyl fluorophosphate
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10 mM, 25% inhibition
additional information
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no inhibition by 10 mM 2-mercaptoethanol, 1 mM iodoacetamide or iodoacetic acid, 10 mM isobutyronitrile or isopropanol
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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molecular imprinting using 2-butanone as additive in the immobilization process improves the synthetic activity of the biocatalyst
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.25
(2R)-2-hydroxy-2-methylbutanenitrile
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pH 5.5, 25°C
2.5
2-hydroxy-2-methylpropanenitrile
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pH 5.5, 25°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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development of an immobilized form of the hydroxynitrile lyase as crosslinked enzyme aggregate (CLEA) with high specific activity (303.5 U/g) and recovery (33%), 180.5 U/g LuCLEA (crosslinked enzyme aggregate) (using sat. (NH4)2SO4), 9.9 U/g LuEA (Enzyme aggregate of LuHNL) (using sat. (NH4)2SO4), 110.9 U/g LuCLEA (using tert-butanol), 221.9 U/g LuEA (using tert-butanol); development of an immobilized form of the hydroxynitrile lyase as crosslinked enzyme aggregate with high specific activity (303.5 U/g) and recovery
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.1 - 5.5
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pH 4.1: about 70% of maximal activity, pH 5.5: maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 4.8
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chromatofocusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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defatted seed meal
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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highest detection level in cytoplasm, with lower levels in organelles, not detected in cell wall or vacuole
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40000
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2 * 40000, SDS-PAGE
42000
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2 * 42000, SDS-PAGE
43000
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2 * 43000, SDS-PAGE
45780
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x * 45780, calculated from sequence
80000
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gel filtration
82000
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gel filtration
87000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 45780, calculated from sequence
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
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half-life: 1 h
5143
5
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immobilization on Eupergit (carrier consisting of macroporous beads) improves the stability considerably in the pH range below pH 5
5143
6 - 11
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stable
5143
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
immobilization on Eupergit (carrier consisting of macroporous beads) improves the stability considerably in the pH range below pH 5
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, stable for at least 45 d
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned into Pichia pastoris, expressed in Escherichia coli as an N-terminal hexa-histidine fusion protein
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cloning of a myc-His-tagged LuHNL-cDNA under control of the methanol-inducible AOX1 (alcohol oxidase) promotor of Pichia pastoris and introduction in the SMD1168 strain. Recombinant LuHNL is kinetically indistinguishable from the authentic flax enzyme
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expressed in Escherichia coli as N-terminal hexa-histidine fusion protein
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expression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G104A
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5-10% of wild-type activity
G95A
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complete destruction of enzymatic activity
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis