(R)-mandelonitrile lyase

 

Involved in cyanogenesis, the release of HCN from injured tissues. Catalyzes the stereospecific addition of HCN to a variety of aldehydes in vitro. Has no oxidase activity. The redox properties of the FAD cofactor appear to be unimportant for catalysis.

 

Reference Protein and Structure

Sequence
Q945K2 UniProt (4.1.2.10) IPR012132 (Sequence Homologues) (PDB Homologues)
Biological species
Prunus dulcis (almond) Uniprot
PDB
3gdp - Hydroxynitrile lyase from almond, monoclinic crystal form (1.57 Å) PDBe PDBsum 3gdp
Catalytic CATH Domains
3.50.50.60 CATHdb 3.30.410.40 CATHdb (see all for 3gdp)
Cofactors
Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:4.1.2.10)

(R)-mandelonitrile
CHEBI:18450ChEBI
→
benzaldehyde
CHEBI:17169ChEBI
+
hydrogen cyanide
CHEBI:18407ChEBI
Alternative enzyme names: (R)-oxynitrilase, D-alpha-hydroxynitrile lyase, D-oxynitrilase, Hydroxynitrile lyase, Oxynitrilase, Mandelonitrile benzaldehyde-lyase,

Enzyme Mechanism

Introduction

The reversible cleavage of alpha-cyanohydrins into HCN and the corresponding aldehyde or ketone follows an ordered Uni Bi mechanism whereby the aldehyde is the first substrate to be bound in the condensation reaction. His497 is proposed to act as a general base abstracting the proton from the mandelonitrile hydroxyl group. This causes the elimination of the cyanide intermediate, which then abstracts a proton from the enzyme.

Catalytic Residues Roles

UniProt PDB* (3gdp)
His486 His459B May act as the general acid/base in the cyanide intermediate protonation step. May also be involved in stabilising the reactive intermediates. electrostatic stabiliser
His524 His497B Acts as a general acid/base. proton acceptor, proton donor
Tyr484, Cys355 Tyr457B, Cys328B Forms a hydrogen bonding network with the hydroxyl/carbonyl group of the substrate. Helps stabilise and active the substrate for the reaction to occur. modifies pKa, electrostatic stabiliser
Lys388 Lys361B Helps activate and stabilise His459. modifies pKa, electrostatic stabiliser
Ser523 Ser496B Helps modify the pKa of the general acid/base histidine. modifies pKa, 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

proton transfer, bimolecular elimination, overall reactant used, overall product formed, native state of enzyme regenerated, inferred reaction step

References

  1. Dreveny I et al. (2009), Biochemistry, 48, 3370-3377. Substrate Binding in the FAD-Dependent Hydroxynitrile Lyase from Almond Provides Insight into the Mechanism of Cyanohydrin Formation and Explains the Absence of Dehydrogenation Activity†,‡. DOI:10.1021/bi802162s. PMID:19256550.
  2. Dreveny I et al. (2001), Structure, 9, 803-815. The hydroxynitrile lyase from almond: a lyase that looks like an oxidoreductase. PMID:11566130.
  3. Jorns MS (1980), Biochim Biophys Acta, 613, 203-209. Studies on the kinetics of cyanohydrin synthesis and cleavage by the the flavoenzyme oxynitrilase. PMID:6246955.

Step 1. His497 abstracts a proton from the substrate, eliminating the cyanide leaving group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys328B modifies pKa
Lys361B modifies pKa
Tyr457B modifies pKa
Ser496B modifies pKa
His459B electrostatic stabiliser
His497B proton acceptor

Chemical Components

proton transfer, ingold: bimolecular elimination, overall reactant used, overall product formed

Step 2. The exact mechanism of this step is still unclear. It is possible that His459 donates a proton to the cyanide intermediate. However, we have shown the proton as coming from His497 in order to regenerate the active site. Water then displaces the products.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys328B electrostatic stabiliser
Lys361B electrostatic stabiliser
Tyr457B electrostatic stabiliser
His459B electrostatic stabiliser
Ser496B electrostatic stabiliser, modifies pKa
His497B proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, overall product formed, inferred reaction step
Download: Image, Marvin File

Step 1. His497 abstracts a proton from the substrate, eliminating the cyanide leaving group.

Step 2. The exact mechanism of this step is still unclear. It is possible that His459 donates a proton to the cyanide intermediate. However, we have shown the proton as coming from His497 in order to regenerate the active site. Water then displaces the products.

Products.

Contributors

Gemma L. Holliday