(S)-hydroxynitrile lyase

 

Hydroxynitrile lyases (HNLs) are important members of the alpha/beta-hydrolase superfamily, and they catalyze the cleavage of cyanohydrins into aldehyde (or ketone) and hydrocyanic acid (HCN). The release of HCN not only protects plant systems from herbivores and microbial attack, but also provides a nitrogen source for the biosynthesis of asparagine.

The enzyme from Hevea brasiliensis tolerates aliphatic (including alpha,beta-unsaturated), aromatic and heterocyclic aldehydes, whereas the enzyme from Manihot esculenta catalyses the addition of hydrogen cyanide to aliphatic, aromatic, heteroaromatic aldehydes and methyl ketones. Both are specific to the S enantiomer. The overall reaction catalysed is very reversible and of importance to synthetic organic chemists.

 

Reference Protein and Structure

Sequence
P52704 UniProt (4.1.2.47) IPR029058 (Sequence Homologues) (PDB Homologues)
Biological species
Hevea brasiliensis (Para rubber tree) Uniprot
PDB
1sc9 - Hydroxynitrile Lyase from Hevea brasiliensis in complex with the natural substrate acetone cyanohydrin (1.8 Å) PDBe PDBsum 1sc9
Catalytic CATH Domains
3.40.50.1820 CATHdb (see all for 1sc9)
Click To Show Structure

Enzyme Reaction (EC:4.1.2.47)

2-hydroxy-2-methylpropanenitrile
CHEBI:15348ChEBI
→
acetone
CHEBI:15347ChEBI
+
hydrogen cyanide
CHEBI:18407ChEBI
Alternative enzyme names: (S)-cyanohydrin producing hydroxynitrile lyase, (S)-oxynitrilase, (S)-HbHNL, (S)-MeHNL, Hydroxynitrile lyase, Oxynitrilase, HbHNL, MeHNL, (S)-selective hydroxynitrile lyase, (S)-cyanohydrin carbonyl-lyase (cyanide forming),

Enzyme Mechanism

Introduction

Although this mechanism utilises the traditional Ser-His-Asp triad of the alpha/beta-hydrolase superfamily, it proceeds via general acid/base catalysis, rather than nucleophilic attack of the serine.

Catalytic Residues Roles

UniProt PDB* (1sc9)
His235 His235A Part of the Ser-His-Asp triad. Acts as a general acid/base, first to deprotonate Ser80, then to donate a proton to the cyanate ion product. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Lys236 Lys236A This residue is instrumental for catalysis in several ways by: correctly positioning the substrate, stabilising the negatively charged reaction product CN(-), and modulating the basicity of the catalytic base. activator, hydrogen bond donor, electrostatic stabiliser, steric role
Asp207 Asp207A Part of the Ser-His-Asp triad, activates the histidine to act as a general acid/base. increase basicity, electrostatic stabiliser, increase acidity
Ser80 Ser80A Part of the Ser-His-Asp triad. Acts as a general acid/base, first donating its proton to His235, and then abstracting a proton from the substrate. proton relay, proton acceptor, electrostatic stabiliser, proton donor
Thr11, Cys81 Thr11A, Cys81A Form part of the oxyanion hole. hydrogen bond donor, electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular elimination, proton transfer, proton relay, native state of enzyme regenerated

References

  1. Zhao Y et al. (2014), Phys Chem Chem Phys, 16, 26864-26875. 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. DOI:10.1039/c4cp04032e. PMID:25375265.
  2. Gartler G et al. (2007), J Biotechnol, 129, 87-97. Structural determinants of the enantioselectivity of the hydroxynitrile lyase from Hevea brasiliensis. DOI:10.1016/j.jbiotec.2006.12.009. PMID:17250917.
  3. Gruber K et al. (2004), J Biol Chem, 279, 20501-20510. Reaction Mechanism of Hydroxynitrile Lyases of the α/β-Hydrolase Superfamily. DOI:10.1074/jbc.m401575200. PMID:14998991.
  4. Lauble H et al. (2001), Acta Crystallogr D Biol Crystallogr, 57, 194-200. Structure of hydroxynitrile lyase fromManihot esculentain complex with substrates acetone and chloroacetone: implications for the mechanism of cyanogenesis. DOI:10.1107/s0907444900015766. PMID:11173464.
  5. Lauble H et al. (2001), Protein Sci, 10, 1015-1022. Mechanistic aspects of cyanogenesis from active-site mutant Ser80Ala of hydroxynitrile lyase from Manihot esculenta in complex with acetone cyanohydrin. DOI:10.1110/ps.01301. PMID:11316882.
  6. Hasslacher M et al. (1996), J Biol Chem, 271, 5884-5891. 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. PMID:8621461.
  7. Wagner UG et al. (1996), Structure, 4, 811-822. Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis. PMID:8805565.

Step 1. His235 (which is present as part of a Ser-His-Asp triad) deprotonates Ser80, deprotonates the hydroxide of the nitrile, initiating an elimination of the cyanate ion.

Binding of the cyanohydrin causes a change in conformation of the Ser80, bringing it into hydrogen bonding distance of the His235. Cleavage of the C-C bond is facilitated by the stabilisation of the evolving negative charge by the positive charge of Lys236, which also shifts the pKa of His235 from 2.5 to 10 [PMID:14998991].

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Catalytic Residues Roles

Residue Roles
Asp207A electrostatic stabiliser
His235A hydrogen bond acceptor, hydrogen bond donor
Thr11A hydrogen bond donor, electrostatic stabiliser
Lys236A hydrogen bond donor, electrostatic stabiliser, activator, steric role
Ser80A proton relay
Cys81A electrostatic stabiliser
Asp207A increase basicity
Ser80A proton acceptor, proton donor
His235A proton acceptor

Chemical Components

ingold: bimolecular elimination, proton transfer, proton relay

Step 2. The cyanate ion deprotonates His235. Upon protonation of the cyanide ion by His235, the hydrogen cyanide is displaced by water. The HCN leaves the active site first, followed by the aldehyde product.

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Catalytic Residues Roles

Residue Roles
Asp207A electrostatic stabiliser
His235A hydrogen bond donor
Thr11A hydrogen bond donor
Lys236A electrostatic stabiliser, hydrogen bond donor, steric role
Thr11A electrostatic stabiliser
Ser80A electrostatic stabiliser
Cys81A electrostatic stabiliser
Asp207A increase acidity
His235A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. His235 (which is present as part of a Ser-His-Asp triad) deprotonates Ser80, deprotonates the hydroxide of the nitrile, initiating an elimination of the cyanate ion.

Binding of the cyanohydrin causes a change in conformation of the Ser80, bringing it into hydrogen bonding distance of the His235. Cleavage of the C-C bond is facilitated by the stabilisation of the evolving negative charge by the positive charge of Lys236, which also shifts the pKa of His235 from 2.5 to 10 [PMID:14998991].

Step 2. The cyanate ion deprotonates His235. Upon protonation of the cyanide ion by His235, the hydrogen cyanide is displaced by water. The HCN leaves the active site first, followed by the aldehyde product.

Products.

Contributors

Gemma L. Holliday, Alex Gutteridge, Craig Porter, James W. Murray