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Information on EC 4.1.2.47 - (S)-hydroxynitrile lyase and Organism(s) Hevea brasiliensis and UniProt Accession P52704
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4.1.2.47 (S)-hydroxynitrile lyaseIUBMB Comments
Hydroxynitrile lyases catalyses the the cleavage of hydroxynitriles into cyanide and the corresponding aldehyde or ketone. In nature the liberation of cyanide serves as a defense mechanism against herbivores and microbial attack in plants. In vitro the enzymes from Manihot esculenta and Hevea brasiliensis accept a broad range of aliphatic and aromatic carbonyl compounds as substrates and catalyse the formation of (S)-hydroxynitriles [1,10].
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Word Map
- 4.1.2.47
- esculenta
- manihot
- brasiliensis
- hevea
- synthesis
- cassava
- ketone
- hcn
- lyases
- benzaldehyde
-
s-mandelonitrile
- rubber
- athnl
- crantz
-
hydrocyanic
-
s-cyanohydrins
-
r-selective
- arylacetonitrilase
-
s-enantiomer
-
cyanogenesis
- montanum
-
s-mandelic
- pharmacology
- agriculture
- analysis
- drug development
- food industry
The taxonomic range for the selected organisms is: Hevea brasiliensis
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
=
+
an aliphatic aldehyde or ketone
Synonyms
mehnl, hbhnl, (s)-hydroxynitrile lyase, alpha-hydroxynitrile lyase, s-hnl, luhnl, s-selective hydroxynitrile lyase, hb-hnl, s-hydroxynitrile lyase, acetone cyanohydrin lyase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
HNL
-
-
Hydroxynitrile lyase
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PATHWAY SOURCE
PATHWAYS
SYSTEMATIC NAME
IUBMB Comments
(S)-cyanohydrin lyase (cyanide forming)
Hydroxynitrile lyases catalyses the the cleavage of hydroxynitriles into cyanide and the corresponding aldehyde or ketone. In nature the liberation of cyanide serves as a defense mechanism against herbivores and microbial attack in plants. In vitro the enzymes from Manihot esculenta and Hevea brasiliensis accept a broad range of aliphatic and aromatic carbonyl compounds as substrates and catalyse the formation of (S)-hydroxynitriles [1,10].
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(2S)-2,3-dimethyl-2-hydroxybutyronitrile
?
binding mode of the chiral substrates is identical to that observed for the biological substrate 2-hydroxy-2-methylpropanenitrile (i.e. acetone cyanohydrin). Three-point binding mode of the substrates: hydrophobic pocket, hydrogen bonds between the hydroxyl group and Ser80 and Thr11, electrostatic interaction of the cyano group with Lys236
-
-
?
(2S)-hydroxy(3-phenoxyphenyl)ethanenitrile
cyanide + 3-phenoxybenzaldehyde
-
-
-
r
cyanide + (2E)-but-2-enal
(3E)-2-hydroxypent-3-enenitrile
-
86% enantiomeric excess with crude enzyme preparation
-
?
cyanide + (2E)-hex-2-enal
(2S,3E)-2-hydroxyhept-3-enenitrile
-
95% enantiomeric excess with crude enzyme preparation
-
?
cyanide + (2Z)-hex-2-enal
(2S,3Z)-2-hydroxyhept-3-enenitrile
-
80% enantiomeric excess with crude enzyme preparation
-
?
cyanide + 1,1'-diformylferrocene
(R,R)-1,1'-bis(cyanohydroxymethyl)ferrocene
a bulky organometallic compound, which does not occur in nature. S-hydroxynitrile lyase from Hevea brasieliensis catalyzes the formation of (R,R)-1,1'-bis(cyanohydroxymethyl)ferrocene at high yield and stereochemical purity
obtained at high yield and stereochemical purity
-
?
cyanide + 2,2-dimethylpropanal
(2S)-2-hydroxy-3,3-dimethylbutanenitrile
-
67% enantiomeric excess
-
?
cyanide + 2-methoxybenzaldehyde
(2S)-2-hydroxy-2-(2-methoxyphenyl)acetonitrile
-
77% enantiomeric excess
-
?
cyanide + 2-methylpropanal
(2S)-2-hydroxy-3-methylbutanenitrile
-
81% enantiomeric excess
-
?
cyanide + 3-methoxybenzaldehyde
(2S)-2-hydroxy-2-(3-methoxyphenyl)acetonitrile
-
99% enantiomeric excess
-
?
cyanide + 3-phenoxybenzaldehyde
(2S)-2-hydroxy-2-(3-phenoxyphenyl)acetonitrile
-
99% enantiomeric excess
-
?
cyanide + 3-phenoxybenzaldehyde
(2S)-hydroxy(3-phenoxyphenyl)acetonitrile
-
20% enantiomeric excess
-
?
cyanide + 3-phenylpropanal
(2S)-2-hydroxy-4-phenylbutanenitrile
-
93% enantiomeric excess
-
?
cyanide + 3-tetrahydrothiophenone
(S)-3-hydroxytetrahydrothiophene-3-carbonitrile
-
-
-
?
cyanide + 4-methoxybenzaldehyde
(2S)-2-hydroxy-2-(4-methoxyphenyl)acetonitrile
-
95% enantiomeric excess
-
?
cyanide + 4-methoxycyclohex-3-ene-1-carbaldehyde
(2S)-hydroxy[4-(methoxy)cyclohex-3-en-1-yl]ethanenitrile
-
-
-
r
cyanide + 4-oxocyclohexanecarbaldehyde
(2S)-hydroxy(4-oxocyclohexyl)ethanenitrile
-
-
-
r
cyanide + 4-[(trimethylsilyl)oxy]cyclohex-3-ene-1-carbaldehyde
(2S)-hydroxy[4-((trimethylsilyl)oxy)cyclohex-3-en-1-yl]ethanenitrile
-
-
-
r
cyanide + benzaldehyde
(2S)-2-hydroxy-2-phenylacetonitrile
-
i.e. (S)-mandelonitrile, more than 99% enantiomeric excess
-
?
cyanide + benzaldehyde
(S)-mandelonitrile
-
94% enantiomeric excess
-
?
cyanide + butanal
(2S)-2-hydroxypentanenitrile
-
80% enantiomeric excess
-
?
cyanide + cinnamaldehyde
(2S)-2-hydroxy-4-phenyl-(E)-but-3-enenitrile
-
95% enantiomeric excess
-
?
cyanide + cyclohex-3-ene-1-carbaldehyde
(2S)-2-(cyclohex-3-enyl)-2-hydroxyacetonitrile
-
99% enantiomeric excess
-
?
cyanide + cyclohexanecarbaldehyde
(2S)-2-cyclohexyl-2-hydroxyacetonitrile
-
99% enantiomeric excess
-
?
cyanide + ferrocenecarboxaldehyde
(R)-(cyanohydroxymethyl)ferrocene
i.e. bis(cyclopentadienyl)iron, a bulky organometallic compound, which does not occur in nature. S-hydroxynitrile lyase from Hevea brasiliensis catalyzes the formation of (R)-(cyanohydroxymethyl)ferrocene at high yield and stereochemical purity
obtained at high yield and stereochemical purity
-
?
cyanide + hexanal
2-hydroxyheptanenitrile
-
84% enantiomeric excess
-
?
cyanide + nonanal
2-hydroxydecanenitrile
-
85% enantiomeric excess
-
?
cyanide + phenylacetaldehyde
(2S)-2-hydroxy-3-phenylpropanenitrile
-
99% enantiomeric excess
-
?
cyanide + prop-2-enal
(2S)-2-hydroxybut-3-enenitrile
-
94% enantiomeric excess with crude enzyme preparation
-
?
cyanide + prop-2-enal
2-hydroxybut-3-enenitrile
-
84% enantiomeric excess
-
?
(1S)-1-furan-2-yl-2-nitroethanol
furan-2-carbaldehyde + CH3NO2
-
enantiomeric excess: ~90%, yield: 60-70%
-
-
r
(1S)-2-nitro-1-(4-nitrophenyl)ethanol
4-nitrobenzaldehyde + CH3NO2
-
enantiomeric excess: ~90%, yield: 60-70%
-
-
r
(1S)-2-nitro-1-phenylethanol
benzaldehyde + CH3NO2
-
enantiomeric excess: ~90%, yield: 60-70%
-
-
r
(1S,2R)-2-nitro-1-phenyl-propanol
benzaldehyde + C2H5NO2
-
4 diastereomers, enantiomeric excess: 95%, yield: 67%
-
-
r
(2S)-1-nitrooctan-2-ol
heptanal + CH3NO2
-
enantiomeric excess: ~90%, yield: 60-70%
-
-
r
(R)-2-(2-furyl)-2-hydroxyacetonitrile
furan-2-carbaldehyde + HCN
-
enantiomeric excess: > 99%, yield: 90%
-
-
r
(S)-2-nitro-1-phenylethanol
nitromethane + benzaldehyde + (R)-2-nitro-1-phenylethanol
-
besides the native cyanohydrins reaction, the enzyme also catalyzes the asymmetric reversible Henry reaction yielding (S)-beta-nitroalcohols with high enantiomeric excess. The catalyst productivity achieved during the resolution is 10times higher than that in the HNL-catalyzed synthesis of (S)-2-nitro-1-phenylethanol
-
-
r
(S)-3-phenoxybenzaldehyde cyanohydrin
m-phenoxybenzaldyhyde + HCN
-
enantiomeric excess: > 98.5%, yield: 95.5%, used for insecticide synthesis
-
-
r
(S)-mandelonitrile
benzaldehyde + HCN
-
enantiomeric excess: > 99%, yield: 90%
-
-
r
3-[(1S)-1-hydroxy-2-nitroethyl]phenol
3-hydroxybenzaldehyde + CH3NO2
-
enantiomeric excess: ~90%, yield: 60-70%
-
-
r
cyanide + (4R)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde
(2S)-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile + (2R)-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile
-
-
the natural substrate benzaldehyde is stereoselectively converted to (R)-mandelonitrile. The non-natural substrate (4R)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde is converted to 47.1% (2S)-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile and 52.9% (2R)-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile
-
?
cyanide + (4R,5S)-2,2,5-trimethyl-1,3-dioxolane-4-carbaldehyde
(2S)-hydroxy-[(4S,5S)-2,2,5-trimethyl-1,3-dioxolan-4-yl]ethanenitrile + (2R)-hydroxy-[(4S,5S)-2,2,5-trimethyl-1,3-dioxolan-4-yl]ethanenitrile
-
-
the natural substrate benzaldehyde is stereoselectively converted to (R)-mandelonitrile. The non-natural substrate (4R,5S)-2,2,5-trimethyl-1,3-dioxolane-4-carbaldehyde is converted to 48.1% (2S)-hydroxy-[(4S,5S)-2,2,5-trimethyl-1,3-dioxolan-4-yl]ethanenitrile and 51.9% (2R)-hydroxy-[(4S,5S)-2,2,5-trimethyl-1,3-dioxolan-4-yl]ethanenitrile
-
?
cyanide + (4R,5S)-2,2-dimethyl-5-phenyl-1,3-dioxolane-4-carbaldehyde
(2S)-[(4S,5S)-2,2-dimethyl-5-phenyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile + (2R)-[(4S,5S)-2,2-dimethyl-5-phenyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile
-
-
the natural substrate benzaldehyde is stereoselectively converted to (R)-mandelonitrile. The non-natural substrate (4R,5S)-2,2-dimethyl-5-phenyl-1,3-dioxolane-4-carbaldehyde is converted to 52.7% (2S)-[(4S,5S)-2,2-dimethyl-5-phenyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile and 47.3% (2R)-[(4S,5S)-2,2-dimethyl-5-phenyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile
-
?
cyanide + (4S)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde
(2S)-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile + (2R)-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile
-
-
the natural substrate benzaldehyde is stereoselectively converted to (R)-mandelonitrile. The non-natural substrate (4S)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde is converted to 34.9% (2S)-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile and 65.1% (2R)-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile
-
?
cyanide + (4S,5R)-2,2,5-trimethyl-1,3-dioxolane-4-carbaldehyde
(2S)-hydroxy-[(4R,5R)-2,2,5-trimethyl-1,3-dioxolan-4-yl]ethanenitrile + (2R)-hydroxy-[(4R,5R)-2,2,5-trimethyl-1,3-dioxolan-4-yl]ethanenitrile
-
-
the natural substrate benzaldehyde is stereoselectively converted to (R)-mandelonitrile. The non-natural substrate (4S,5R)-2,2,5-trimethyl-1,3-dioxolane-4-carbaldehyde is converted to 35.1% (2S)-hydroxy-[(4R,5R)-2,2,5-trimethyl-1,3-dioxolan-4-yl]ethanenitrile and 64.9% (2R)-hydroxy-[(4R,5R)-2,2,5-trimethyl-1,3-dioxolan-4-yl]ethanenitrile
-
?
cyanide + (4S,5R)-2,2-dimethyl-5-phenyl-1,3-dioxolane-4-carbaldehyde
(2S)-[(4R,5R)-2,2-dimethyl-5-phenyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile + (2R)-[(4R,5R)-2,2-dimethyl-5-phenyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile
-
-
the natural substrate benzaldehyde is stereoselectively converted to (R)-mandelonitrile. The non-natural substrate (4S,5R)-2,2-dimethyl-5-phenyl-1,3-dioxolane-4-carbaldehyde is converted to 49.9% (2S)-[(4R,5R)-2,2-dimethyl-5-phenyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile and 50.1% (2R)-[(4R,5R)-2,2-dimethyl-5-phenyl-1,3-dioxolan-4-yl](hydroxy)ethanenitrile
-
?
cyanide + 1,4-dioxaspiro[4.5]decane-2-carbaldehyde
(S)-2-hydroxy-2-((R)-1,4-dioxaspiro[4.5]decan-2-yl)acetonitrile + (R)-2-hydroxy-2-((R)-1,4-dioxaspiro[4.5]decan-2-yl)acetonitrile + (S)-2-hydroxy-2-((S)-1,4-dioxaspiro[4.5]decan-2-yl)acetonitrile + (R)-2-hydroxy-2-((S)-1,4-dioxaspiro[4.5]decan-2-yl)acetonitrile
-
-
the natural substrate benzaldehyde is stereoselectively converted to (R)-mandelonitrile. The non-natural substrate 1,4-dioxaspiro[4.5]decane-2-carbaldehyde is converted to 16.9% (2S)-(2R)-1,4-dioxaspiro[4.5]dec-2-yl(hydroxy)ethanenitrile, 33.0% (2R)-(2R)-1,4-dioxaspiro[4.5]dec-2-yl(hydroxy)ethanenitrile, 18.3% (2S)-(2S)-1,4-dioxaspiro[4.5]dec-2-yl(hydroxy)ethanenitrile and 31.8% (2R)-(2S)-1,4-dioxaspiro[4.5]dec-2-yl(hydroxy)ethanenitrile
-
?
cyanide + tetrahydro-2H-pyran-2-carbaldehyde
(2S)-hydroxy-[(2R)-tetrahydro-2H-pyran-2-yl]ethanenitrile + (2R)-hydroxy-[(2R)-tetrahydro-2H-pyran-2-yl]ethanenitrile + (2S)-hydroxy-[(2S)-tetrahydro-2H-pyran-2-yl]ethanenitrile + (2R)-hydroxy-[(2S)-tetrahydro-2H-pyran-2-yl]ethanenitrile
-
-
the natural substrate benzaldehyde is stereoselectively converted to (R)-mandelonitrile. The non-natural substrate tetrahydro-2H-pyran-2-carbaldehyde is converted to 5.4% (2S)-hydroxy-[(2R)-tetrahydro-2H-pyran-2-yl]ethanenitrile, 45.9% (2R)-hydroxy-[(2R)-tetrahydro-2H-pyran-2-yl]ethanenitrile, 3.9% (2S)-hydroxy[(2S)-tetrahydro-2H-pyran-2-yl]ethanenitrile and 44.9% (2R)-hydroxy[(2S)-tetrahydro-2H-pyran-2-yl]ethanenitrile
-
?
cyanide + tetrahydrofuran-2-carbaldehyde
(2S)-hydroxy-[(2R)-tetrahydrofuran-2-yl]ethanenitrile + (2R)-hydroxy-[(2R)-tetrahydrofuran-2-yl]ethanenitrile + (2S)-hydroxy-[(2S)-tetrahydrofuran-2-yl]ethanenitrile + (2R)-hydroxy-[(2S)-tetrahydrofuran-2-yl]ethanenitrile
-
-
the natural substrate benzaldehyde is stereoselectively converted to (R)-mandelonitrile. The non-natural substrate tetrahydrofuran-2-carbaldehyde is converted to 17.1% (2S)-hydroxy-[(2R)-tetrahydrofuran-2-yl]ethanenitrile, 32.9% (2R)-hydroxy-[(2R)-tetrahydrofuran-2-yl]ethanenitrile, 18.9% (2S)-hydroxy[(2S)-tetrahydrofuran-2-yl]ethanenitrile and 31.1% (2R)-hydroxy[(2S)-tetrahydrofuran-2-yl]ethanenitrile
-
?
cyanide + thiophene-2-carbaldehyde
(2S)-hydroxy(thiophen-2-yl)ethanenitrile
-
-
-
-
?
HCN + (benzyloxy)acetaldehyde
3-(benzyloxy)-(2S)-2-hydroxy-propanenitrile + 3-(benzyloxy)-(2R)-2-hydroxy-propanenitrile
-
-
50% 3-(benzyloxy)-(2S)-2-hydroxy-propanenitrile and 50% 3-(benzyloxy)-(2R)-2-hydroxy-propanenitrile
?
HCN + 1,1'-diformylferrocene
(R,R)-1,1-bis(cyanohydroxymethyl)ferrocene
-
-
-
?
HCN + 2-methyldihydrofuran
3-hydroxy-2-methyltetrahydrofuran-3-carbonitrile
-
analysis of diastereomeric distribution of the products, depending on different reaction conditions such as pH, reaction time, and solvent properties
-
?
HCN + 2-methyldihydrothiophen-3(2H)-one
3-hydroxy-2-methyltetrahydrothiophen-3-carbonitrile
-
analysis of diastereomeric distribution of the products, depending on different reaction conditions such as pH, reaction time, and solvent properties
-
?
HCN + 2-naphthaldehyde
(2S)-2-hydroxynaphthalen-2-yl-acetonitrile + (2R)-2-hydroxynaphthalen-2-yl-acetonitrile
-
-
83% (2S)-2-hydroxynaphthalen-2-yl-acetonitrile and 17% (2R)-2-hydroxynaphthalen-2-yl-acetonitrile
?
HCN + 2-naphthylacetaldehyde
(2S)-2-hydroxy-3-naphthalen-1-yl-propionitrile + (2R)-2-hydroxy-3-naphthalen-1-yl-propionitrile
-
-
84.3% (2S)-2-hydroxy-3-naphthalen-1-yl-propionitrile and 15.6% (2R)-2-hydroxy-3-naphthalen-1-yl-propionitrile
?
HCN + 3-phenoxybenzaldehyde
(2S)-hydroxy(3-phenoxyphenyl)acetonitrile
-
-
-
?
HCN + 3-phenoxypropanal
(2S)-2-hydroxy-4-phenoxybutanenitrile + (2S)-2-hydroxy-4-phenoxybutanenitrile
-
-
95.8% (2S)-2-hydroxy-4-phenoxybutanenitrile and 4.2% (2R)-2-hydroxy-4-phenoxybutanenitrile
?
HCN + 3-phenylpropionaldehyde
(2S)-2-hydroxy-4-phenylbutanenitrile
-
-
89% enantiomeric excess
?
HCN + acrolein
(2S)-2-hydroxybut-3-enenitrile
-
-
92% enantiomeric ecxess
?
HCN + cyclohexanecarbaldehyde
(2R)-cyclohexyl(hydroxy)acetonitrile
-
-
-
?
HCN + decanal
(S)-2-hydroxyundecanenitrile
-
reaction in a two phase solvent system aqueous buffer and ionic liquid. Compared to the use of organic solvents as the nonaqueous phase, the reaction rate is significantly increased whereas the enantioselectivity remains good
-
-
?
HCN + dodecanal
(S)-2-hydroxytridecanenitrile
-
reaction in a two phase solvent system aqueous buffer and ionic liquid. Compared to the use of organic solvents as the nonaqueous phase, the reaction rate is significantly increased whereas the enantioselectivity remains good
-
-
?
HCN + furaldehyde
(2R)-furan-2-yl(hydroxy)ethanenitrile
-
enzyme encapsulated in sol-gel matrix
-
-
?
HCN + hexanal
(S)-2-hydroxyoctanenitrile
-
enzyme encapsulated in sol-gel matrix
-
-
?
HCN + m-phenoxybenzaldehyde
(S)-hydroxy-(3-phenoxy-phenyl)acetonitrile
-
enzyme encapsulated in sol-gel matrix
-
-
?
HCN + methyl isopropyl ketone
(S)-2-hydroxy-2,3-dimethylbutanenitrile
-
enzyme encapsulated in sol-gel matrix
-
-
?
HCN + phenylacetaldehyde
(2R)-2-hydroxy-3-phenylpropanenitrile
-
-
-
?
HCN + tetrahydro-2H-3-pyranone
(3R)-3-hydroxytetrahydro-2H-pyran-3-carbonitrile
-
-
enantiomeric excess at pH 4.75 is 48.3%
?
HCN + tetrahydro-3-furanone
(3R)-3-hydroxytetrahydrofuran-3-carbonitrile
-
-
81% enantiomeric excess
?
HCN + undecanal
(S)-2-hydroxydodecanenitrile
-
reaction in a two phase solvent system aqueous buffer and ionic liquid. Compared to the use of organic solvents as the nonaqueous phase, the reaction rate is significantly increased whereas the enantioselectivity remains good
-
-
?
(S)-mandelonitrile
cyanide + benzaldehyde
binding mode of the chiral substrate is identical to that observed for the biological substrate acetone cyanohydrin. Three-point binding mode of the substrates: hydrophobic pocket, hydrogen bonds between the hydroxyl group and Ser80 and Thr11, electrostatic interaction of the cyano group with Lys236
-
-
?
(S)-mandelonitrile
cyanide + benzaldehyde
enzyme kinetics in both directions is studied on a model system with mandelonitrile, benzaldehyde, and HCN using two different methods: initial rate measurements and progress curve analysis. Ordered Uni bi mechanism with the formation of a dead-end complex of enzyme, (S)-mandelonitrile and HCN. HCN is the first product released from the enzyme followed by benzaldehyde while in the synthesis reaction, benzaldehyde is the first substrate bond to the enzyme followed by HCN
-
-
r
(S)-mandelonitrile
cyanide + benzaldehyde
the enzymatic reversible conversion of (S)-mandelonitrile to HCN and benzaldehyde can be adequately described by a three-step, reversible-ordered UniBi reaction scheme
-
-
r
(S)-mandelonitrile
cyanide + benzaldehyde
the enzyme is a member of the alpha/beta hydrolase fold protein family, containing a catalytic triad with C-C cleaving and ligating activity
-
-
r
2-hydroxy-2-methylpropanenitrile
cyanide + acetone
i.e. acetone cyanohydrin, three-point binding mode of the substrates: hydrophobic pocket, hydrogen bonds between the hydroxyl group and Ser80 and Thr11, electrostatic interaction of the cyano group with Lys236
-
-
?
2-furaldehyde cyanohydrin
2-furaldehyde + HCN
-
aqua gel, 30 min, conversion ratio: 89%, enantiomeric excess: 94%
-
-
r
2-furaldehyde cyanohydrin
2-furaldehyde + HCN
-
free enzyme, 30 min, conversion ratio: 89%,enantiomeric excess: 94%
-
-
r
2-hydroxy-2-methylpropanenitrile
acetone + HCN
-
release of HCN serves as a defense against herbivores and microbial attack of the plant
-
?
2-hydroxy-2-methylpropanenitrile
HCN + acetone
-
liberation of HCN by degradation of acetone cyanohydrin is considerably faster than the consumption of HCN by the formation of (S)-mandelonitrile
-
?
2-hydroxy-2-methylpropanenitrile
HCN + acetone
-
release of HCN serves as a defense against herbivores and microbial attack of the plant
-
?
HCN + benzaldehyde
(S)-mandelonitrile
-
-
99% enantiomeric excess
?
HCN + benzaldehyde
(S)-mandelonitrile
-
enzyme encapsulated in sol-gel matrix
-
-
?
HCN + benzaldehyde
(S)-mandelonitrile
-
reaction in a two phase solvent system aqueous buffer and ionic liquid. Compared to the use of organic solvents as the nonaqueous phase, the reaction rate is significantly increased whereas the enantioselectivity remains good
-
-
?
hexanal cyanohydrin
hexanal + HCN
-
aqua gel, 2 h, conversion ratio: 92%, enantiomeric excess: 94%
-
-
r
hexanal cyanohydrin
hexanal + HCN
-
free enzyme, 4 h, conversion ratio: 91%,enantiomeric excess: 94%
-
-
r
m-phenoxybenzaldehyde cyanohydrin
m-phenoxybenzaldehyde + HCN
-
aqua gel, 72 h, conversion ratio: 92%, enantiomeric excess: 98%
-
-
r
m-phenoxybenzaldehyde cyanohydrin
m-phenoxybenzaldehyde + HCN
-
free enzyme, 72 h, conversion ratio: 45%,enantiomeric excess: 82%
-
-
r
mandelonitrile
benzaldehyde + HCN
-
aqua gel, 0.5 h, conversion ratio: 97%, enantiomeric excess: 99%
-
-
r
mandelonitrile
benzaldehyde + HCN
-
CLEA, 72 h, conversion ratio: 55%, enantiomeric excess: 67%
-
-
r
mandelonitrile
benzaldehyde + HCN
-
free enzyme, 4 h, conversion ratio: 97%,enantiomeric excess: 97%
-
-
r
additional information
?
-
(3E)-2-hydroxy-4-phenylbut-3-enenitrile is not sufficently accepted by the enzyme in crude enzyme preparation
-
-
?
additional information
?
-
modeling of the complexes of the enzyme with its natural substrate acetone cyanohydrin as well as with the chiral compounds mandelonitrile and 2,3-dimethyl-2-hydroxybutyronitril. Enzymatic mechanism involves catalytic triad Ser80, His235, and Asp207 as a genertal acid/base
-
-
?
additional information
?
-
-
modeling of the complexes of the enzyme with its natural substrate acetone cyanohydrin as well as with the chiral compounds mandelonitrile and 2,3-dimethyl-2-hydroxybutyronitril. Enzymatic mechanism involves catalytic triad Ser80, His235, and Asp207 as a genertal acid/base
-
-
?
additional information
?
-
-
theoretical investigation of the catalytic mechanism
-
-
?
additional information
?
-
theoretical investigation of the catalytic mechanism
-
-
?
additional information
?
-
on the basis of extensive QM/MM MD and RAMD MD simulations, the catalytic mechanism of the enzyme and its substrate delivery and product (HCN) release are explored. The catalytic reaction approximately follows a two-stage mechanism. The first stage involves two fast processes including the proton abstraction of the substrate through a double-proton transfer and the C-CN bond cleavage, while the second stage concerns HCN formation and is rate-determining
-
-
?
additional information
?
-
-
acetylferrocene and 1,1'-diacetylferrocene are not transformed with this enzyme
-
-
?
additional information
?
-
-
acetylferrocene and 1,1'-diacetylferrocene are not transformed with this enzyme
-
?
additional information
?
-
-
the enantiomeric excess of the product is optimal at pH 5.4 and at HCN concentration between 200 mM and 400 mM and clearly decreases at concentrations greater than 1.5 M. When the temperature decreases from 25°C to -5°C, the enantiomeric excess increases from 88% to 95%
-
-
?
additional information
?
-
-
the enantiomeric excess of the product is optimal at pH 5.4 and at HCN concentration between 200 mM and 400 mM and clearly decreases at concentrations greater than 1.5 M. When the temperature decreases from 25°C to -5°C, the enantiomeric excess increases from 88% to 95%
-
?
additional information
?
-
-
the enzyme catalyzes asymmetric cyanohydrin and Henry reactions. (R)-2-nitro-1-phenylethanol is not a substrate or HNL
-
-
?
additional information
?
-
-
the enzyme has low activity in performing Henry reactions
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2-hydroxy-2-methylpropanenitrile
acetone + HCN
-
release of HCN serves as a defense against herbivores and microbial attack of the plant
-
?
2-hydroxy-2-methylpropanenitrile
HCN + acetone
-
release of HCN serves as a defense against herbivores and microbial attack of the plant
-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
benzaldehyde
acts as a linear competitive inhibitor against mandelonitrile
benzaldehyde
-
strong competitive inhibitor, the half-life of HNL in 20 mM citrate-phosphate buffer (pH 5.0) in the presence of 10 mM of benzaldehyde is around 50 min compared to 450 min in the absence of benzaldehyde
additional information
PMSF, NEM or 4-amidinophenylmethanesulfonylfluoride do not inhibit enzymatic activity
-
additional information
-
PMSF, NEM or 4-amidinophenylmethanesulfonylfluoride do not inhibit enzymatic activity
-
additional information
-
(R)-2-nitro-1-phenylethanol and nitromethane have negligible inhibition effects on HNL activity
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
dibutyl ether
-
best solvent for HCN concentrations around 300 mM HCN concentration
additional information
-
reaction in a two phase solvent system aqueous buffer and ionic liquid. Compared to the use of organic solvents as the nonaqueous phase, the reaction rate is significantly increased whereas the enantioselectivity remained good
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.2
(S)-mandelonitrile
-
in 50 mM citrate buffer (pH 5.0), temperature not specified in the publication
0.4
2-nitro-1-phenylethanol
mutant enzyme F125T/L146M, pH and temperature not specified in the publication
0.7
2-nitro-1-phenylethanol
mutant enzyme F125T/Y133F, pH and temperature not specified in the publication
1.4
2-nitro-1-phenylethanol
mutant enzyme V106F, pH and temperature not specified in the publication
1.6
2-nitro-1-phenylethanol
mutant enzyme V106F/L121Y, pH and temperature not specified in the publication
1.8
2-nitro-1-phenylethanol
mutant enzyme V106F/F125T, pH and temperature not specified in the publication
5
2-nitro-1-phenylethanol
mutant enzyme C81L, pH and temperature not specified in the publication
5
2-nitro-1-phenylethanol
mutant enzyme F125T, pH and temperature not specified in the publication
5
2-nitro-1-phenylethanol
mutant enzyme L121Y, pH and temperature not specified in the publication
5
2-nitro-1-phenylethanol
mutant enzyme L121Y/F125T/L146M, pH and temperature not specified in the publication
5
2-nitro-1-phenylethanol
mutant enzyme L121Y/F125T/Y133F, pH and temperature not specified in the publication
5
2-nitro-1-phenylethanol
mutant enzyme L121Y/L146M, pH and temperature not specified in the publication
5
2-nitro-1-phenylethanol
mutant enzyme L146M, pH and temperature not specified in the publication
5
2-nitro-1-phenylethanol
mutant enzyme V106F/L121Y/F125T, pH and temperature not specified in the publication
5
2-nitro-1-phenylethanol
mutant enzyme V106F/L146M, pH and temperature not specified in the publication
6.1
2-nitro-1-phenylethanol
wild type enzyme, pH and temperature not specified in the publication
7.1
2-nitro-1-phenylethanol
mutant enzyme Y133F, pH and temperature not specified in the publication
0.6
mandelonitrile
mutant enzyme L152F, pH and temperature not specified in the publication
0.7
mandelonitrile
mutant enzyme I209G, pH and temperature not specified in the publication
0.8
mandelonitrile
mutant enzyme I209A, pH and temperature not specified in the publication
1.4
mandelonitrile
mutant enzyme V106F/L121Y, pH and temperature not specified in the publication
1.5
mandelonitrile
mutant enzyme F125T/L146M, pH and temperature not specified in the publication
2
mandelonitrile
mutant enzyme F125T, pH and temperature not specified in the publication
2
mandelonitrile
mutant enzyme V106F/L121Y/F125T, pH and temperature not specified in the publication
2.1
mandelonitrile
mutant enzyme F125T/Y133F, pH and temperature not specified in the publication
2.7
mandelonitrile
mutant enzyme L121Y, pH and temperature not specified in the publication
3.1
mandelonitrile
mutant enzyme L121Y/F125T, pH and temperature not specified in the publication
3.1
mandelonitrile
mutant enzyme Y133F, pH and temperature not specified in the publication
3.3
mandelonitrile
wild type enzyme, pH and temperature not specified in the publication
3.6
mandelonitrile
mutant enzyme L121Y/F125T/L146M, pH and temperature not specified in the publication
3.7
mandelonitrile
mutant enzyme L146M, pH and temperature not specified in the publication
5.3
mandelonitrile
mutant enzyme V106F, pH and temperature not specified in the publication
6.7
mandelonitrile
mutant enzyme L121Y/L146M, pH and temperature not specified in the publication
8.6
mandelonitrile
mutant enzyme F210I, pH and temperature not specified in the publication
8.6
mandelonitrile
mutant enzyme L148F, pH and temperature not specified in the publication
9.4
mandelonitrile
mutant enzyme C81L, pH and temperature not specified in the publication
0.4
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, mutant enzyme F125T/L146M
0.7
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, mutant enzyme F125T/Y133F
1.4
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, mutant enzyme V106F
1.6
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, mutant enzyme V106F/L121Y
1.8
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, mutant enzyme V106F/F125T
6.1
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, wild-type enzyme
7.1
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, mutant enzyme Y133F
0.6
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L152F
0.7
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme I209G
0.8
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme I209A
1.4
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme V106F/L121Y
1.5
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme F125T/L146M
1.5
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121F
2
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme F125T
2
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme V106F/L121Y/F125T
2.1
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme F125T/Y133F
2.7
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121T
2.7
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Y
2.8
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121A
3.1
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/F125T
3.1
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme Y133F
3.3
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, wild-type enzyme
3.6
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/F125T/L146M
3.7
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121V
3.7
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L146M
4
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121M
4.9
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121I
5.3
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme V106F
5.6
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121W
5.7
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Q
6.6
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121S
6.7
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/L146M
8
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121H
8.1
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121C
8.6
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme F210I
8.6
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L148F
9.4
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme C81L
2.6
(S)-2-nitro-1-phenylethanol
-
in 50 mM phosphate buffer pH 6.0, at 25°C
2.6
(S)-2-nitro-1-phenylethanol
-
in 50 mM phosphate buffer pH 6.0, at 22°C
additional information
additional information
a kinetic investigation based on the rate curve method
-
additional information
additional information
-
a kinetic investigation based on the rate curve method
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.83
(S)-mandelonitrile
-
in 50 mM citrate buffer (pH 5.0), temperature not specified in the publication
0.1
2-nitro-1-phenylethanol
mutant enzyme F125T/L146M, pH and temperature not specified in the publication
0.1
2-nitro-1-phenylethanol
mutant enzyme L121Y/L146M, pH and temperature not specified in the publication
0.1
2-nitro-1-phenylethanol
mutant enzyme V106F, pH and temperature not specified in the publication
0.1
2-nitro-1-phenylethanol
mutant enzyme V106F/L121Y, pH and temperature not specified in the publication
0.12
2-nitro-1-phenylethanol
mutant enzyme F125T/Y133F, pH and temperature not specified in the publication
0.15
2-nitro-1-phenylethanol
wild type enzyme, pH and temperature not specified in the publication
0.2
2-nitro-1-phenylethanol
mutant enzyme C81L, pH and temperature not specified in the publication
0.2
2-nitro-1-phenylethanol
mutant enzyme V106F/F125T, pH and temperature not specified in the publication
0.2
2-nitro-1-phenylethanol
mutant enzyme V106F/L146M, pH and temperature not specified in the publication
0.3
2-nitro-1-phenylethanol
mutant enzyme Y133F, pH and temperature not specified in the publication
0.5
2-nitro-1-phenylethanol
mutant enzyme L121Y, pH and temperature not specified in the publication
0.5
2-nitro-1-phenylethanol
mutant enzyme L121Y/F125T/L146M, pH and temperature not specified in the publication
0.5
2-nitro-1-phenylethanol
mutant enzyme L121Y/F125T/Y133F, pH and temperature not specified in the publication
0.5
2-nitro-1-phenylethanol
mutant enzyme L146M, pH and temperature not specified in the publication
0.5
2-nitro-1-phenylethanol
mutant enzyme V106F/L121Y/F125T, pH and temperature not specified in the publication
0.6
2-nitro-1-phenylethanol
mutant enzyme F125T, pH and temperature not specified in the publication
3
mandelonitrile
mutant enzyme I209G, pH and temperature not specified in the publication
7
mandelonitrile
mutant enzyme L152F, pH and temperature not specified in the publication
7
mandelonitrile
mutant enzyme V106F, pH and temperature not specified in the publication
17
mandelonitrile
mutant enzyme L148F, pH and temperature not specified in the publication
20
mandelonitrile
mutant enzyme L146M, pH and temperature not specified in the publication
21
mandelonitrile
mutant enzyme F210I, pH and temperature not specified in the publication
22
mandelonitrile
mutant enzyme Y133F, pH and temperature not specified in the publication
23
mandelonitrile
mutant enzyme I209A, pH and temperature not specified in the publication
25
mandelonitrile
wild type enzyme, pH and temperature not specified in the publication
32
mandelonitrile
mutant enzyme F125T/L146M, pH and temperature not specified in the publication
32
mandelonitrile
mutant enzyme F125T/Y133F, pH and temperature not specified in the publication
33
mandelonitrile
mutant enzyme C81L, pH and temperature not specified in the publication
43
mandelonitrile
mutant enzyme L121Y/F125T, pH and temperature not specified in the publication
45
mandelonitrile
mutant enzyme L121Y/F125T/L146M, pH and temperature not specified in the publication
55
mandelonitrile
mutant enzyme V106F/L121Y, pH and temperature not specified in the publication
66
mandelonitrile
mutant enzyme F125T, pH and temperature not specified in the publication
82
mandelonitrile
mutant enzyme V106F/L121Y/F125T, pH and temperature not specified in the publication
106
mandelonitrile
mutant enzyme L121Y, pH and temperature not specified in the publication
0.1
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, mutant enzyme F125T/L146M
0.1
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, mutant enzyme V106F
0.12
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, mutant enzyme F125T/Y133F
0.15
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, wild-type enzyme
0.2
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, mutant enzyme V106F/F125T
0.3
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, mutant enzyme Y133F
0.6
rac-2-nitro-1-phenylethanol
pH 5.0, temperature not specified in the publication, mutant enzyme F125T
3
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme I209G
3
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121T
5
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121H
6
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Q
7
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L152F
7
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme V106F
9
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121S
15
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121V
15
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121W
17
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L148F
20
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121C
20
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L146M
21
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme F210I
22
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme Y133F
23
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme I209A
23
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121A
25
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, wild-type enzyme
32
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme F125T/L146M
32
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme F125T/Y133F
33
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme C81L
33
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121I
43
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121M
43
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/F125T
45
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/F125T/L146M
55
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme V106F/L121Y
66
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme F125T
77
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121F
82
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme V106F/L121Y/F125T
106
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Y
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.55
(S)-mandelonitrile
-
in 50 mM citrate buffer (pH 5.0), temperature not specified in the publication
1
mandelonitrile
mutant enzyme L148F, pH and temperature not specified in the publication
1.4
mandelonitrile
mutant enzyme V106F, pH and temperature not specified in the publication
2.5
mandelonitrile
mutant enzyme F210I, pH and temperature not specified in the publication
3.5
mandelonitrile
mutant enzyme C81L, pH and temperature not specified in the publication
4
mandelonitrile
mutant enzyme I209G, pH and temperature not specified in the publication
5.4
mandelonitrile
mutant enzyme L146M, pH and temperature not specified in the publication
7.1
mandelonitrile
mutant enzyme Y133F, pH and temperature not specified in the publication
7.4
mandelonitrile
wild type enzyme, pH and temperature not specified in the publication
11
mandelonitrile
mutant enzyme L152F, pH and temperature not specified in the publication
12
mandelonitrile
mutant enzyme L121Y/F125T/L146M, pH and temperature not specified in the publication
14
mandelonitrile
mutant enzyme L121Y/F125T, pH and temperature not specified in the publication
15
mandelonitrile
mutant enzyme F125T/Y133F, pH and temperature not specified in the publication
21
mandelonitrile
mutant enzyme F125T/L146M, pH and temperature not specified in the publication
29
mandelonitrile
mutant enzyme I209A, pH and temperature not specified in the publication
33
mandelonitrile
mutant enzyme F125T, pH and temperature not specified in the publication
39
mandelonitrile
mutant enzyme L121Y, pH and temperature not specified in the publication
39
mandelonitrile
mutant enzyme V106F/L121Y, pH and temperature not specified in the publication
41
mandelonitrile
mutant enzyme V106F/L121Y/F125T, pH and temperature not specified in the publication
0.6
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121H
1
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L148F
1.1
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Q
1.4
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121S
1.4
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121T
1.4
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme V106F
2.5
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme F210I
2.5
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121C
2.6
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121W
3.5
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme C81L
4
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme I209G
4
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121V
5.4
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L146M
6.6
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121I
7.1
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme Y133F
7.4
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, wild-type enzyme
8.3
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121A
11
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121M
11
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L152F
12
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/F125T/L146M
14
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/F125T
15
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme F125T/Y133F
21
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme F125T/L146M
29
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme I209A
33
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme F125T
39
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121Y
39
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme V106F/L121Y
41
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme V106F/L121Y/F125T
51
rac-mandelonitrile
pH 5.0, temperature not specified in the publication, mutant enzyme L121F
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.1
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121P
0.13
0.15
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme C81L
0.17
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme V106F
0.2
0.21
0.27
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme F125T
0.29
0.32
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme L146M
0.39
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme V106F/F125T
0.56
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/F125T/Y133F
0.62
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme L121Y
0.7
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121N
0.71
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/F125T/L146M
0.9
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme I12A
1.4
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121G
115
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/L146M
119
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121F
12
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L152F
14
140
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme V106F/L121Y/F125T
149
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121Y
16
2
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121T
22
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme C81L
24
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L146M
28
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme Y133F
3.8
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121S
30
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121A
33
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, wild-type enzyme
35
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121I
4
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121H
42
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme I209A
5.1
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme I209G
5.9
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121Q
52
53
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme F125T/L146M
55
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/F125T
6.7
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme V106F
64
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/F125T/L146M
7
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L148F
88
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme V106F/L121Y
92
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme F125T
additional information
0.13
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme L121Y/L146M
0.13
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, wild-type enzyme
0.2
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme F125T/L146M
0.2
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121E
0.21
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme F125T/Y133F
0.21
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme V106F/L146M
0.29
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme V106F/L121Y/F125T
0.29
substrate: rac-2-nitro-1-phenylethanol, pH 5.0, temperature not specified in the publication, mutant enzyme Y133F
14
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme F210I
14
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121W
16
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121C
16
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121V
52
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme F125T/Y133F
52
substrate: rac-mandelonitrile, pH 5.0, temperature not specified in the publication, mutant enzyme L121M
additional information
screening assay for hydroxynitrile lyases and its application in high-throughput screening of Escherichia coli mutant libraries, semi-quantitative test where the rate of colour formation corresponds to the particular enzyme activity of the sample
additional information
-
expressed in Saccharomyces cerevisiae or Pichia pastoris
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
-
the maximal activity of HNL in (S)-2-nitro-1-phenylethanol cleavage is at/above pH 7.5
additional information
additional information
-
to reduce chemical background reaction, pH-values below 5 are necessary
additional information
-
to reduce chemical background reaction, pH-values below 5 are necessary
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
-5 - 25
-
when the temperature decreases from 25°C to -5°C, the enantiomeric excess increases from 88% to 95%
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
hydroxynitrile lyases defend plants from herbivores and microbial attack by releasing cyanide from hydroxynitriles
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). HNL_HEVBR
257
0
29228
Swiss-Prot
Secretory Pathway (Reliability: 4)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homotetramer
-
4 * 29200, in solution, calculated from amino acid sequence
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structure of the hydroxynitrile lyase at 1.9 A resolution. The structure belongs to the alpha/beta hydrolase superfamily. Its active site is deeply buried inside the protein, and connected to the outside by a narrow tunnel. The catalytic triade consists of residues Ser80, His235 and Asp207. By analogy with other alpha/beta hydrolases, the oxyanion hole is formed by the mainchain-NH of Cys81 and by the side-chains of Cys81 and Thr11
hanging drop vapor diffusion method, freeze-quench method to prepare crystals of the complex of the enzyme with acetone cyanohydrin, complex of mutant K236L enzyme with acetone, structure of the K236L with acetone cyanohydrin-acetone cyanohydrin shows the substrate in a different orientation from the wild-type complex
hanging drop vapor diffusion. crystal structures of hydroxynitrile lyase: one native and three complexes with acetone, isopropyl alcohol, and thiocyanate
hanging-drop vapor-diffusion method. The crystals belong to the orthorhombic space group C222(1) with cell dimensions of a = 47.6, b = 106.8 and c = 128.2 A. The crystals diffract to about 2.5 A resolution on a rotating-anode X-ray source
vapor diffusion hanging drop method. X-ray crystal structures (at 1.54 and 1.76 A resolution) of HbHNL complexes with two chiral substrates (S)-mandelonitrile and (2S)-2,3-dimethyl-2-hydroxybutyronitrile by soaking and rapid freeze quenching techniques. Only the S-enantiomers of the two substrates are observed in the active site
crystals obtained by sitting drop vapor diffusion, space group C222(1), unit cell dimensions a = 47.29 A, b = 106.66 A, c = 128.16 A
-
enzyme in complex with acetone, hexafluoroacetone, trichloroacetaldehyde or rhodanine, hanging-drop vapour diffusion method, space group C222(1)
-
three-dimensional structure analysis, catalytic triad consisting of serine, histidine and aspartic acid
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C81L
C81S
E79A
F125T
F125T/L146M
the mutant shows higher specific activity towards racemic mandelonitrile compared to the wild type enzyme
F125T/Y133F
the mutant shows higher specific activity towards racemic mandelonitrile compared to the wild type enzyme
F210I
H103L/W128A
increased activity with the substrate (2S)-hydroxy[4-(methoxy)cyclohex-3-en-1-yl]ethanenitrile compared to the starting clone W128A
H10A
mutation results in a 30000 Da protein with increased electrophoretic mobility on native high percentage (16%) polyacrylamide gels. the mutant enzyme displays almost wild-type specific activity in crude extracts, suggesting that His10 is not crucial for activity. However, activity is almost completely lost during purification, supporting the possibility that the H10A exchange has a destabilizing effect and may prevent formation of an active dimer of the enzyme after purification
H235A
I12A
mutant yields mostly insoluble protein, which suggests that the substitution hinders protein folding
I209A
I209G
K236L
inactive mutant protein, three-dimensional structure is similar to wild-type enzyme
L121F
6.9fold increase in specificity constants (kcat/Km) for racemic mandelonitrile
L121Y
L121Y/F125T
L121Y/F125T/L146M
L121Y/L146M
the mutant shows higher specific activity towards racemic mandelonitrile compared to the wild type enzyme
L146M
the mutant shows lower specific activity towards racemic mandelonitrile compared to the wild type enzyme
L148F
the mutant shows lower specific activity towards racemic mandelonitrile compared to the wild type enzyme
L152F
S80A
V106F
the mutant shows lower specific activity towards racemic mandelonitrile compared to the wild type enzyme
V106F/L121Y
the mutant shows higher specific activity towards racemic mandelonitrile compared to the wild type enzyme
V106F/L121Y/F125T
W128A
W128A/I219V
very weak activity in cleavage reaction with (2S)-hydroxy[4-(methoxy)cyclohex-3-en-1-yl]ethanenitrile
W128A/K147R
very weak activity in cleavage reaction with (2S)-hydroxy[4-(methoxy)cyclohex-3-en-1-yl]ethanenitrile
W128A/P187L
no activity in cleavage reaction with (2S)-hydroxy[4-(methoxy)cyclohex-3-en-1-yl]ethanenitrile
W128A/Q215H
slightly increased activity with the substrate (2S)-hydroxy[4-(methoxy)cyclohex-3-en-1-yl]ethanenitrile compared to the starting clone W128A
Y133F
the mutant shows lower specific activity towards racemic mandelonitrile compared to the wild type enzyme
C81L
the mutant has high enantioselectivity towards (R)-mandelonitrile with a kcat value 1.3times higher compared to the wild type enzyme
C81S
no difference can be observed in the electrophoretic mobilities between the wild-type and mutant protein on native polyacrylamide gels and by isoelectric focusing. Mutation does not change the charge or size of the amino acid side chain, but nevertheless greatly reduces activity
E79A
no difference can be observed in the electrophoretic mobilities between the wild-type and mutant protein on native polyacrylamide gels and by isoelectric focusing. Mutation greatly reduces, but does not abolish activity. The negative charge provided by Glu-79 may be required in the active site, but a direct participation of this residue in enzyme catalysis is not suggested
F125T
2.6fold increase in kcat for racemic mandelonitrile, 4.5fold increase in specificity constants (kcat/Km) for racemic mandelonitrile
F125T
the mutant has high enantioselectivity towards (R)-mandelonitrile with a kcat value 2.6times and a kcat/Km value 4.5times higher compared to the wild type enzyme
F210I
mutant yields mostly insoluble protein, which suggests that the substitution hinders protein folding
F210I
the mutant shows lower specific activity towards racemic mandelonitrile compared to the wild type enzyme
H235A
inactive mutant enzyme, no difference can be observed in the electrophoretic mobilities between the wild-type and mutant protein on native polyacrylamide gels and by isoelectric focusing
I209A
3.9fold increase in specificity constants (kcat/Km) for racemic mandelonitrile
I209A
the mutant has higher enantioselectivity towards (R)-mandelonitrile with a kcat/Km value 3.9times higher compared to the wild type enzyme
I209G
mutant yields mostly insoluble protein, which suggests that the substitution hinders protein folding, moderate substrate inhibition by rac-mandelonitrile
I209G
the mutant shows lower specific activity towards racemic mandelonitrile compared to the wild type enzyme
L121Y
5.3fold increase in specificity constants (kcat/Km) for racemic mandelonitrile, 4.2fold increase in kcat for racemic mandelonitrile, the mutant enzyme shows the same high (S)-enantioselectivity (98% ee) as wild-type enzyme. The mutant catalyzes the cleavage of 2-nitro-1-phenylethanol 4.8 times more efficiently than the wild type enzyme, this is similar to the 4.5-fold increase seen for mandelonitrile cleavage
L121Y
the mutant has high enantioselectivity towards (S)-mandelonitrile with a kcat value 4.2times higher and a kcat/Km value 5.3times higher compared to the wild type enzyme
L121Y/F125T
1.7fold increase in kcat for racemic mandelonitrile, 1.9fold increase in specificity constants (kcat/Km) for racemic mandelonitrile
L121Y/F125T
the mutant has high enantioselectivity towards (R)-mandelonitrile with a kcat value 4.2times higher and a kcat/Km value 1.7times higher compared to the wild type enzyme
L121Y/F125T
the mutant shows higher specific activity towards racemic mandelonitrile compared to the wild type enzyme
L121Y/F125T/L146M
5.5fold increase in activity over the wild type enzyme
L121Y/F125T/L146M
the mutant shows higher specific activity towards racemic mandelonitrile compared to the wild type enzyme
L152F
1.5fold increase in specificity constants (kcat/Km) for racemic mandelonitrile
L152F
the mutant has higher enantioselectivity towards (R)-mandelonitrile with a kcat/Km value 1.5times higher compared to the wild type enzyme
S80A
no difference can be observed in the electrophoretic mobilities between the wild-type and mutant protein on native polyacrylamide gels and by isoelectric focusing
V106F/L121Y/F125T
3.3fold increase in kcat for racemic mandelonitrile, 5.5fold increase in specificity constants (kcat/Km) for racemic mandelonitrile
V106F/L121Y/F125T
the mutant has high enantioselectivity towards (R)-mandelonitrile with a kcat value 3.3times higher compared to the wild type enzyme
W128A
higher conversion and better selectivity than wild-type enzyme with the substrate 4-methoxycyclohex-3-ene-1-carbaldehyde
W128A
lower conversion and lower selectivity than wild-type enzyme with the substrate 4-methoxycyclohex-3-ene-1-carbaldehyde
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3.5
enzyme is inactivated very fast. Stability decreases with higher buffer concentrations at pH 3.5 in all three buffer systems (phosphate buffer, glutamate buffer, citrate buffer)
705188
4
decativation below pH 4.0, an unfolding of the enzyme followed by aggregation leading to closely packed enzyme particles at low pH values
703489
6.5
very stable above. At pH 6.5 stability increases with increasing buffer concentration in all three systems (phosphate buffer, glutamate buffer, citrate buffer). The enzyme is most stable in phosphate buffer whereas for glutamate buffer the relative increase of half-life with concentration is more pronounced
705188
5
-
unstable below. Fivefold increase in half-life under optimal conditions by Venoruton and monohydroxyethylrutoside. 50% increase in half-life by 5-20 ng/ml rutin and 1.5-6 ng/ml by hyperoside
655659
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
half-life: 1755 min in phosphate buffer, 1379 min in glutamate buffer, 2315 min in citrate buffer
40
half-life: 690 min in phosphate buffer, 204 min in glutamate buffer, 322 min in citrate buffer
50
half-life: 342 min in phosphate buffer, 80 min in glutamate buffer, 115 min in citrate buffer
60
half-life: 56 min in phosphate buffer, 42 min in glutamate buffer, 29 min in citrate buffer
70
half-life: 10 min in phosphate buffer, 15 min in glutamate buffer, 7 min in citrate buffer
40 - 50
-
the enzyme shows a half-life time of 411 h at 40°C. At 50°C a rapid inactivation is observed
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
half-life at pH 5 and 20°C is halved at a concentration of 2 mM benzaldehyde and 30 mM HCN, stability decreases drastically with increasing concentrations of benzaldehyde
-
half-life increases linearly with enzyme concentration in the aqueous buffer as well as in a two-phase system of buffer and dibutyl ether
-
influence of different organic solvents and solvent mixtures on the stability in two-phase systems of aqueous buffer and organic solvent
-
the enzyme shows especially high stability in mixtures of methyl-tert-butylether and hexane, 40:60 and 25:75
-
the use of solvent mixtures with methyl-tert-butylether leads to better stability than the use of pure solvents
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hexane
-
inactivates within a few min, the use of solvent mixtures with methyl-tert-butylether leads to better stability than the use of pure solvents the enzyme shows especially high stability in mixtures of methyl-tert-butylether and hexane, 40:60 and 25:75
methyl-tert-butylether
-
the use of solvent mixtures with methyl-tert-butylether leads to better stability than the use of pure solvents the enzyme shows especially high stability in mixtures of methyl-tert-butylether and hexane, 40:60 and 25:75
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
unstable below pH 5, fivefold increase in half-life under optimal conditions by Venoruton and monohydroxyethylrutoside, 50% increase in half-life by 5-20 ng/ml rutin and 1.5-6 ng/ml by hyperoside
-
651390, 655659
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
mutant enzymes Hnl-E79A, Hnl-S80A, Hnl-C81S, and Hnl-H235A and the wild-type protein are purified to homogeneity, and Hnl-H10A is partially purified, by ion exchange chromatography and native polyacrylamide gel electrophoresis
Ni-NTA agarose column chromatography and Sephadex G-75 gel filtration
preparation of a cross-linked enzyme aggregate (CLEA) from hydroxynitrile lyase from Hevea brasiliensis, precipitated hydroxynitrile lyase, comparison of free, cross-linked enzyme aggregate (CLEA) and immobilized in sol-gel (aqua gel)
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of mutant and wild-type proteins in Saccharomyces cerevisiae
expression of wild-type and mutant enzymes in Escherichia coli BL21
limitations in enzyme supply from natural resources are overcome by production of the enzyme in the microbial host systems Escherichia coli, Sachycharomyces cerevisiae, and Pichia pastoris. Expression of Hnl in the prokaryotic system leads to the formation of inclusion bodies whereas in both yeast hosts high levels of soluble protein are obtained. Highest yields are obtained in a high cell density batch fermentation of a Pichia pastoris transformant that expresses heterologous Hnl to about 50% of the soluble cytosolic protein. At a cell density of 100 g/liter cell dry weight, a volume yield of 22 g/liter of heterologous product is obtained. Attempts to produce the Hnl protein extracellularly with the yeast hosts by applying different leader peptide strategies are not successful. Immunofluorescence microscopy studies indicate that the secretion-directed heterologous Hnl protein accumulates in the plasma membrane forming aggregated clusters of inactive protein
cloned and produced by heterologous expression in different microbial hosts
-
full-length cDNA functinal expressed in Escherichia coli, Pichia pastoris and Saccharomyces cerevisiae
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
agriculture
-
enantiomerically pure cyanohydrins produced by enzyme-catalyzed synthesis are important synthetic intermediates for agrochemicals
pharmacology
-
enantiomerically pure cyanohydrins produced by enzyme-catalyzed synthesis are important synthetic intermediates for pharmaceuticals
synthesis
synthesis
the cyanohydrin reaction of formylferrocene catalysed by the hydroxynitrile lyase from Hevea brasiliensis provides an access to chiral ferrocene derivatives in high enantiomeric excess. Since cyanohydrins are versatile synthetic intermediates, the possibility for many preparative transformations is opened. This synthetic potential is enlarged even further with the transformation of 1,1'-diformylferrocene leading to biscyanohydrins
synthesis
the enzyme is a potent biocatalyst for the industrial production of chemicals
synthesis
-
biocatalyst for the enantiospecific addition of hydrogen cyanide to aldehydes in organic solvents
synthesis
-
catalyzes the industrially interesting formation of (S)-cyanohydrins from aldehydes or ketones and HCN
synthesis
-
industrial important biocatalyst used in enantiospecific syntheses of alpha-hydroxynitriles from aldehydes and methyl-ketones
synthesis
-
production of chiral ferrocene derivatives as ligands in asymmetric catalysis, to bioelectrochemistry and development of new pharmaceuticals against malaria
synthesis
-
crude cell lysate containing a hydroxynitrile lyase can be used for the enantioselective synthesis of several cyanohydrins in a microchannel. These enzymatic reactions show a high initial reaction rate and enantioselectivity, which in a batchwise process can only be achieved by vigorous stirring
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Selmar, D.; Lieberei, R.; Biehl, B.; Conn, E.E.
alpha-Hydroxynitrile lyase in Hevea brasiliensis and its significance for rapid cyanogenesis
Physiol. Plant.
75
97-101
1989
Hevea brasiliensis
-
Wagner, U.G.; Hasslacher, M.; Griengl, H.; Schwab, H.; Kratky, C.
Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis
Structure
4
811-822
1996
Hevea brasiliensis (P52704)
Effenberger, F.
Synthesis and reactions of optically active cyanohydrins
Angew. Chem. Int. Ed. Engl.
33
1555-1564
1994
Hevea brasiliensis
-
Klempier, N.; Griengl, H.
Alphatic (S)-cyanohydrins by enzyme catalyzed synthesis
Tetrahedron Lett.
34
4769-4772
1993
Hevea brasiliensis (P52704)
-
Hasslacher, M.; Schall, M.; Hayn, M.; Bona, R.; Rumbold, K.; Lueckl, J.; Griengl, H.; Kohlwein, S.D.; Schwab, H.
High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts
Protein Expr. Purif.
11
61-71
1997
Hevea brasiliensis (P52704), Hevea brasiliensis
Wajant, H.; Foerster, S.
Purification and characterization of hydroxynitrile lyase from Hevea brasiliensis
Plant Sci.
115
25-31
1996
Hevea brasiliensis (P52704)
-
Hasslacher, M.; Schall, M.; Hayn, M.; Griengl, H.; Kohlwein, S.D.; Schwab, H.
(S)-Hydroxynitrile lyase from Hevea brasiliensis
Ann. N. Y. Acad. Sci.
799
707-712
1996
Hevea brasiliensis
Hanefeld, U.; Stranzl, G.; Straathof, A.J.; Heijnen, J.J.; Bergmann, A.; Mittelbach, R.; Glatter, O.; Kratky, C.
Electrospray ionization mass spectrometry, circular dichroism and SAXS studies of the (S)-hydroxynitrile lyase from Hevea brasiliensis
Biochim. Biophys. Acta
1544
133-142
2001
Hevea brasiliensis
Gruber, K.; Gugganig, M.; Wagner, U.G.; Kratky, C.
Atomic resolution crystal structure of hydroxynitrile lyase from Hevea brasiliensis
Biol. Chem.
380
993-1000
1999
Hevea brasiliensis
Effenberger, F.
Enzyme-catalyzed preparation and synthetic applications of optically active cyanohydrins
Chimia
53
3-10
1999
Hevea brasiliensis
-
Bauer, M.; Griengl, H.; Steiner, W.
Parameters influencing stability and activity of a S-hydroxynitrile lyase from Hevea brasiliensis in two-phase systems
Enzyme Microb. Technol.
24
514-522
1999
Hevea brasiliensis
-
Costes, D.; Wehtje, E.; Adlercreutz, P.
Hydroxynitrile lyase-catalyzed synthesis of cyanohydrins in organic solvents. Parameters influencing activity and enantiospecificity
Enzyme Microb. Technol.
25
384-391
1999
Hevea brasiliensis, Manihot esculenta
-
Geyer, R.; Kartnig, T.; Griengl, H.; Steiner, W.
Influence of flavonoids on the stability of an (S)-hydroxynitrile lyase from Hevea brasiliensis
Food. Technol. Biotechnol.
39
161-167
2001
Hevea brasiliensis
-
Stranzl, G.R.; Gruber, K.; Steinkellner, G.; Zangger, K.; Schwab, H.; Kratky, C.
Observation of a short, strong hydrogen bond in the active site of hydroxynitrile lyase from Hevea brasiliensis explains a large pKa shift of the catalytic base induced by the reaction intermediate
J. Biol. Chem.
279
3699-3707
2004
Hevea brasiliensis (P52704), Hevea brasiliensis
Zuegg, J.; Gruber, K.; Gugganig, M.; Wagner, U.G.; Kratky, C.
Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis
Protein Sci.
8
1990-2000
1999
Hevea brasiliensis
Gruber, K.
Elucidation of the mode of substrate binding to hydroxynitrile lyase from Hevea brasiliensis
Proteins
44
26-31
2001
Hevea brasiliensis (P52704), Hevea brasiliensis
Froehlich, R.F.G.; Zabelinskaja-Mackova, A.A.; Fechter, M.H.; Griengl, H.
Novel access to chiral 1,1'-disubstituted ferrocene derivatives via double stereoselective cyanohydrin synthesis exploiting the hydroxynitrile lyase from Hevea brasiliensis
Tetrahedron
14
355-362
2003
Hevea brasiliensis
-
Bianchi, P.; Roda, G.; Riva, S.; Danieli, B.; Zabelinskaja-Mackova, A.; Griengl, H.
On the selectivity of oxynitrilases towards alpha-oxygenated aldehydes
Tetrahedron
57
2213-2220
2001
Hevea brasiliensis
-
Avi, M.; Fechter, M.J.; Gruber, K.; Belaj, F.; Pchlauer, P.; Griengl, H.
Hydroxynitrile lyase catalysed synthesis of heterocyclic (R)- and (S)-cyanohydrins
Tetrahedron
60
10411-10418
2004
Hevea brasiliensis
-
Griengl, H.; Schwab, H.; Fechter, M.
The synthesis of chiral cyanohydrins by oxynitrilases
Trends Biotechnol.
18
252-256
2000
Hevea brasiliensis, Manihot esculenta
Geyer, R.; Kartnig, T.; Griengl, H.; Steiner, W.
Influence of flavonoids on the stability of an (S)-hydroxynitrile lyase from Hevea brasiliensis
Food Technol. Biotechnol.
39
161-167
2001
Hevea brasiliensis
-
Gruber, K.; Gartler, G.; Krammer, B.; Schwab, H.; Kratky, C.
Reaction mechanism of hydroxynitrile lyases of the alpha/beta-hydrolase superfamily: The three-dimensional structure of the transient enzyme-substrate complex certifies the crucial role of LYS236
J. Biol. Chem.
279
20501-20510
2004
Hevea brasiliensis (P52704), Hevea brasiliensis
Yeow, Y.L.; Leong, Y.K.; Cheah, M.Y.; Dang, H.D.; Law, C.K.
Cleaving of S-mandelonitrile catalyzed by S-hydroxynitrile lyase from Hevea brasiliensis--a kinetic investigation based on the rate curve method
J. Biotechnol.
111
31-39
2004
Hevea brasiliensis (P52704), Hevea brasiliensis
Gaisberger, R.P.; Fechter, M.H.; Griengl, H.
The first hydroxynitrile lyase catalysed cyanohydrin formation in ionic liquids
Tetrahedron
15
2959-2963
2004
Hevea brasiliensis
-
Veum, L.; Hanefeld, U.; Pierre, A.
The first encapsulation of hydroxynitrile lyase from Hevea brasiliensis in a sol-gel matrix
Tetrahedron
60
10419-10425
2004
Hevea brasiliensis
-
Cabirol, F.L.; Hanefeld, U.; Sheldon, R.A.
Immobilized hydroxynitrile lyases for enantioselective synthesis of cyanohydrins: sol-gels and cross-linked enzyme aggregates
Adv. Synth. Catal.
348
1645-1654
2006
Hevea brasiliensis
-
Purkarthofer, T.; Skranc, W.; Schuster, C.; Griengl, H.
Potential and capabilities of hydroxynitrile lyases as biocatalysts in the chemical industry
Appl. Microbiol. Biotechnol.
76
309-320
2007
Hevea brasiliensis
Krammer, B.; Rumbold, K.; Tschemmernegg, M.; Poechlauer, P.; Schwab, H.
A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening
J. Biotechnol.
129
151-161
2007
Hevea brasiliensis (P52704)
Koch, K.; van den Berg, R.J.; Nieuwland, P.J.; Wijtmans, R.; Wubbolts, M.G.; Schoemaker, H.E.; Rutjes, F.P.; van Hest, J.C.
Enzymatic synthesis of optically pure cyanohydrins in microchannels using a crude cell lysate
Chem. Eng. J.
135
S89-S92
2008
Hevea brasiliensis
-
Avi, M.; Wiedner, R.M.; Griengl, H.; Schwab, H.
Improvement of a stereoselective biocatalytic synthesis by substrate and enzyme engineering: 2-hydroxy-(4-oxocyclohexyl)acetonitrile as the model
Chemistry
14
11415-11422
2008
Hevea brasiliensis (P52704)
Schmidt, A.; Gruber, K.; Kratky, C.; Lamzin, V.S.
Atomic resolution crystal structures and quantum chemistry meet to reveal subtleties of hydroxynitrile lyase catalysis
J. Biol. Chem.
283
21827-21836
2008
Hevea brasiliensis (P52704)
Ueberbacher, B.J.; Griengl, H.; Weber, H.
Chemo-enzymatic synthesis of new ferrocenyl-oxazolidinones and their application as chiral auxiliaries
Tetrahedron Asymmetry
19
838-846
2008
Hevea brasiliensis (P52704)
-
Wagner, U.G.; Schall, M.; Hasslacher, M.; Hayn, M.; Griengl, H.; Schwab, H.; Kratky, C.
Crystallization and preliminary X-ray diffraction studies of a hydroxynitrile lyase from Hevea brasiliensis
Acta Crystallogr. Sect. D
52
591-593
1996
Hevea brasiliensis (P52704)
Bauer, M.; Griengl, H.; Steiner, W.
Kinetic studies on the enzyme (S)-hydroxynitrile lyase from Hevea brasiliensis using initial rate methods and progress curve analysis
Biotechnol. Bioeng.
62
20-29
1999
Hevea brasiliensis (P52704), Hevea brasiliensis
Fechter, M.H.; Gruber, K.; Avi, M.; Skranc, W.; Schuster, C.; Pchlauer, P.; Klepp, K.O.; Griengl, H.
Stereoselective biocatalytic synthesis of (S)-2-hydroxy-2-methylbutyric acid via substrate engineering by using "thio-disguised" precursors and oxynitrilase catalysis
Chemistry
13
3369-3376
2007
Hevea brasiliensis (P52704)
Hickel, A.; Graupner, M.; Lehner, D.; Hemnetter, A.; Glatter, O.; Griengl, H.
Stability of the hydroxynitrile lyase from Hevea brasiliensis: a fluorescence and dynamic light scattering study
Enzyme Microb. Technol.
21
361-366
1997
Hevea brasiliensis (P52704)
-
Hasslacher, M.; Schall, M.; Hayn, M.; Griengl, H.; Kohlwein, S.D.; Schwab, H.
Molecular cloning of the full-length cDNA of (S)-hydroxynitrile lyase from Hevea brasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue
J. Biol. Chem.
271
5884-5891
1996
Hevea brasiliensis (P52704), Hevea brasiliensis
Gartler, G.; Kratky, C.; Gruber, K.
Structural determinants of the enantioselectivity of the hydroxynitrile lyase from Hevea brasiliensis
J. Biotechnol.
129
87-97
2007
Hevea brasiliensis (P52704), Hevea brasiliensis
Guterl, J.K.
Andexer, J.N.; Sehl, T.; von Langermann, J.; Frindi-Wosch, I.; Rosenkranz, T.; Fitter, J.; Gruber, K.; Kragl, U.; Eggert, T.; Pohl, M.: Uneven twins: comparison of two enantiocomplementary hydroxynitrile lyases with alpha/beta-hydrolase fold
J. Biotechnol.
141
166-173
2009
Hevea brasiliensis, Manihot esculenta, Manihot esculenta (P52705)
Bauer, M.; Geyer, R.; Boy, M.; Griegl, G.; Steiner, W.
Stability of the enzyme (S)-hydroxynitrile lyase from Hevea brasiliensis
J. Mol. Catal. B
5
343-347
1998
Hevea brasiliensis (P52704)
-
Cui, F.C.; Pan, X.L.; Liu, J.Y.
Catalytic mechanism of hydroxynitrile lyase from Hevea brasiliensis: a theoretical investigation
J. Phys. Chem. B
114
9622-9628
2010
Hevea brasiliensis, Hevea brasiliensis (P52704)
Hasslacher, M.; Kratky, C.; Griengl, H.; Schwab, H.; Kohlwein, S.D.
Hydroxynitrile lyase from Hevea brasiliensis: molecular characterization and mechanism of enzyme catalysis
Proteins
27
438-449
1997
Hevea brasiliensis (P52704), Hevea brasiliensis
Frhlich, R.F.G.; Zabelinskaja-Mackova, A.A.; Fechter, M.H.; Griengl, H.
Novel access to chiral 1,1'-disubstituted ferrocene derivatives via double stereoselective cyanohydrin synthesis exploiting the hydroxynitrile lyase from Hevea brasiliensis
Tetrahedron Asymmetry
14
355-362
2003
Hevea brasiliensis (P52704)
-
Klempier, N.; Pichler, U.; Griengl, H.
Synthesis of alpha,beta-unsaturated (S)-cyanohydrins using the oxynitrilase from Hevea brasiliensis
Tetrahedron Asymmetry
6
845-848
1995
Hevea brasiliensis (P52704)
-
Schmidt, M.; Herve, S.; KLempier, N.; Griengl, H.
Preparation of optically active cyanohydrins using the (S)-hydroxynitrile lyase from Hevea brasiliensis
Tetrahedron
52
7833-7840
1996
Hevea brasiliensis (P52704)
-
Yuryev, R.; Purkarthofer, T.; Gruber, M.; Griengl, H.; Liese, A.
Kinetic studies of the asymmetric Henry reaction catalyzed by hydroxynitrile lyase from Hevea brasiliensis
Biocatal. Biotransform.
28
348-356
2010
Hevea brasiliensis
-
Yuryev, R.; Briechle, S.; Gruber-Khadjawi, M.; Griengl, H.; Liese, A.
Asymmetric retro-Henry reaction catalyzed by hydroxynitrile lyase from Hevea brasiliensis
ChemCatChem
2
981-986
2010
Hevea brasiliensis
-
Padhi, S.K.; Fujii, R.; Legatt, G.A.; Fossum, S.L.; Berchtold, R.; Kazlauskas, R.J.
Switching from an esterase to a hydroxynitrile lyase mechanism requires only two amino acid substitutions
Chem. Biol.
17
863-871
2010
Hevea brasiliensis, Manihot esculenta
von Langermann, J.; Nedrud, D.M.; Kazlauskas, R.J.
Increasing the reaction rate of hydroxynitrile lyase from Hevea brasiliensis toward mandelonitrile by copying active site residues from an esterase that accepts aromatic esters
ChemBioChem
15
1931-1938
2014
Hevea brasiliensis (P52704), Hevea brasiliensis
Rauwerdink, A.; Lunzer, M.; Devamani, T.; Jones, B.; Mooney, J.; Zhang, Z.J.; Xu, J.H.; Kazlauskas, R.J.; Dean, A.M.
Evolution of a catalytic mechanism
Mol. Biol. Evol.
33
971-979
2016
Hevea brasiliensis
Zhao, Y.; Chen, N.; Mo, Y.; Cao, Z.
A full picture of enzymatic catalysis by hydroxynitrile lyases from Hevea brasiliensis Protonation dependent reaction steps and residue-gated movement of the substrate and the product
Phys. Chem. Chem. Phys.
16
26864-26875
2014
Hevea brasiliensis (P52704)
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