Mandelonitrile Lyase
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The
enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
(''R'')-mandelonitrile lyase (, ''(R)-HNL'', ''(R)-oxynitrilase'', ''(R)-hydroxynitrile lyase'') catalyzes the
chemical reaction A chemical reaction is a process that leads to the chemistry, chemical transformation of one set of chemical substances to another. When chemical reactions occur, the atoms are rearranged and the reaction is accompanied by an Gibbs free energy, ...
:mandelonitrile \rightleftharpoons hydrogen cyanide + benzaldehyde This enzyme belongs to the family of
lyase In biochemistry, a lyase is an enzyme that catalyzes the breaking (an elimination reaction) of various chemical bonds by means other than hydrolysis (a substitution reaction) and oxidation Redox ( , , reduction–oxidation or oxidatio ...
s, specifically the aldehyde-lyases, which cleave carbon-carbon bonds. The
systematic name A systematic name is a name given in a systematic way to one unique group, organism, object or chemical substance, out of a specific population or collection. Systematic names are usually part of a nomenclature. A semisystematic name or semitrivi ...
of this enzyme class is mandelonitrile benzaldehyde-lyase (hydrogen cyanide-forming). Other names in common use include hydroxynitrile lyase, (R)-oxynitrilase, oxynitrilase, D-oxynitrilase, D-alpha-hydroxynitrile lyase, and mandelonitrile benzaldehyde-lyase. This enzyme participates in cyanoamino acid metabolism. It has 2 cofactors: flavin, and
flavoprotein Flavoproteins are proteins that contain a nucleic acid derivative of riboflavin. These proteins are involved in a wide array of biological processes, including removal of radicals contributing to oxidative stress, photosynthesis, and DNA repair. ...
.


Historical perspective

Mandelonitrile lyases, more colloquially referred to as HNLs (hydroxynitrile lyases) were first characterized by Wöhler in 1938, based on their high activity in almond. Since then, HNLs have been isolated from a wide variety of plants including stone fruits, sorghum grains, millipedes, and passion fruits. HNLs are peculiar in that, within the same organism and even the same sample, there exist a variety of different
isoforms A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene and are the result of genetic differences. While many perform the same or similar biological roles, some isoforms have uniqu ...
of this enzyme. These isoforms are not able to be determined from one another based on factors influencing activity. This variety also results from macro-heterogeneity, as some isoforms bind
FAD A fad, trend, or craze is any form of collective behavior that develops within a culture, a generation, or social group in which a group of people enthusiastically follow an impulse for a short time period. Fads are objects or behaviors tha ...
at their N-terminus while others are unable to bind FAD. It is understood that this is the case because the N-terminal fold is a region known to bind FAD as a needed cofactor. Also curious is that FAD plays no observed role in active site oxidation-reduction reactions of this enzyme. Those HNLs that bind FAD do so at a
hydrophobic In chemistry, hydrophobicity is the chemical property of a molecule (called a hydrophobe) that is seemingly repelled from a mass of water. In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, thu ...
region neighboring the active site where it is believed that the binding of FAD confers structural stability that allows for enzymatic action. These HNL, referred to as HNL Class I (or HNL I) are also noted to have N-terminus
glycosylation Glycosylation is the reaction in which a carbohydrate (or ' glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. In biology (but not ...
and the distinct heterogeneity and presence of isoforms within the same organism. HNL Class II (HNL II), on the other hand, afford a wider variety of substrates, and in general favor (S)
stereochemistry Stereochemistry, a subdiscipline of chemistry, studies the spatial arrangement of atoms that form the structure of molecules and their manipulation. The study of stereochemistry focuses on the relationships between stereoisomers, which are defined ...
, whereas HNL I stereo-selectively produce (R)-mandelonitrile.


Structure and action

Due to the simple purification of this enzyme (5-30 fold purification is sufficient to reach homogeneity), its biological and biochemical analysis have been very thoroughly studied. In addition to the study of many
isoforms A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene and are the result of genetic differences. While many perform the same or similar biological roles, some isoforms have uniqu ...
within a given organism, there has been study dedicated to the understanding of HNL localization, the physical structure of the enzyme and its active site, and the mechanisms by which it is able to mediate this important set of reactions. Upon the purification of Black Cherry HNL, research from Wu and Poulton raised
antiserum In immunology, antiserum is a blood serum containing antibodies (either monoclonal or polyclonal) that is used to spread passive immunity to many diseases via blood donation ( plasmapheresis). For example, convalescent serum, or passive ant ...
to these specific HNL, which were then applied (with colloidal gold particles in tow) to Black Cherry
cotyledon A cotyledon ( ; ; "a cavity, small cup, any cup-shaped hollow", gen. (), ) is a "seed leaf" – a significant part of the embryo within the seed of a plant – and is formally defined as "the embryonic leaf in seed-bearing plants, one or mor ...
and
endosperm The endosperm is a tissue produced inside the seeds of most of the flowering plants following double fertilization. It is triploid (meaning three chromosome sets per nucleus) in most species, which may be auxin-driven. It surrounds the Embryo#Pla ...
. Here it was found that HNL overwhelmingly localizes to the cell walls of these developing plants. It was so enriched in these regions that it was noted upwards of 5% of the
cell wall A cell wall is a structural layer that surrounds some Cell type, cell types, found immediately outside the cell membrane. It can be tough, flexible, and sometimes rigid. Primarily, it provides the cell with structural support, shape, protection, ...
images taken via
Electron Microscopy An electron microscope is a microscope that uses a beam of electrons as a source of illumination. It uses electron optics that are analogous to the glass lenses of an optical light microscope to control the electron beam, for instance focusing i ...
imaged the gold particles that were indirectly labelling these proteins. Knowing where this protein is highly localized, Figure 1 details work that highlights the structure of this protein and the residues in its active site respectively. Of specific interest, HNLs make use of a catalytically active Cys residue. While
Cysteine Cysteine (; symbol Cys or C) is a semiessential proteinogenic amino acid with the chemical formula, formula . The thiol side chain in cysteine enables the formation of Disulfide, disulfide bonds, and often participates in enzymatic reactions as ...
residues are conserved throughout species in three separate locations (at the
N-terminal The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...
FAD binding site, and two at the
C-terminal The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When t ...
active site), it appears that the catalytically active residue lies near the active site, suggesting an important role in HNL catalytic action. Other structural features indicative of HNL are split based on their class. While Class II HNL are known to be more heterogenous and more often seen in
grain A grain is a small, hard, dry fruit (caryopsis) – with or without an attached husk, hull layer – harvested for human or animal consumption. A grain crop is a grain-producing plant. The two main types of commercial grain crops are cereals and ...
s, Class I HNL are more typically FAD-binding and function as seed storage proteins. This action allows for increased amino acid metabolism in developing seeds. Because the enzyme is able to quickly reverse this reaction to create
hydrogen cyanide Hydrogen cyanide (formerly known as prussic acid) is a chemical compound with the chemical formula, formula HCN and structural formula . It is a highly toxic and flammable liquid that boiling, boils slightly above room temperature, at . HCN is ...
, HNLs play an essential role in defense of the seed As of late 2007, only one
structure A structure is an arrangement and organization of interrelated elements in a material object or system, or the object or system so organized. Material structures include man-made objects such as buildings and machines and natural objects such as ...
has been solved for this class of enzymes, with the PDB accession code .


Mechanism of action


HNLs are known to be stereospecific, giving the action of this enzyme a major advantage in effectively creating precursors essential to the metabolic development of
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...
s and a wide range of clinically relevant small molecules. The wide variety of organisms and isoforms that constitute the HNL family however, has been determined to yield a variety of different mechanisms that facilitate this reaction in a stereospecific way. Figures 2 and 3 detail the typical synthetic and solved biochemical mechanisms for the formation of this key metabolic intermediate. Key differences between these pathways rely mostly on the lack of enantiomeric specificity conferred through the synthetic pathways despite the use of similar classes of reactions. In addition, most of the synthetic methods for facilitating this set of reactions take place in
organic solvent A solvent (from the Latin '' solvō'', "loosen, untie, solve") is a substance that dissolves a solute, resulting in a solution. A solvent is usually a liquid but can also be a solid, a gas, or a supercritical fluid. Water is a solvent for p ...
, whereas it has been shown that HNL activity is highest at a polar-nonpolar interface.


Disease relevance

HNLs and the action they mediate is a key target for study of protein engineering, as the formation of mandelonitrile is a key step in a wide variety of organic syntheses with medical and therapeutic potential. The step mediated by these enzymes is essential to the synthesis of stereospecific bond formation in (R)-Salbutamol
bronchodilator A bronchodilator or broncholytic (although the latter occasionally includes secretory inhibition as well) is a substance that dilates the bronchi and bronchioles, decreasing resistance in the respiratory airway and increasing airflow to the lun ...
s, (S)-
amphetamine Amphetamine (contracted from Alpha and beta carbon, alpha-methylphenethylamine, methylphenethylamine) is a central nervous system (CNS) stimulant that is used in the treatment of attention deficit hyperactivity disorder (ADHD), narcolepsy, an ...
s, (1R, 2S)-(-)-
ephedrine Ephedrine is a central nervous system (CNS) stimulant and sympathomimetic agent that is often used to prevent hypotension, low blood pressure during anesthesia. It has also been used for asthma, narcolepsy, and obesity but is not the preferred ...
bronchodilators, in addition to many others, including Lipitor,
Thalidomide Thalidomide, sold under the brand names Contergan and Thalomid among others, is an oral administered medication used to treat a number of cancers (e.g., multiple myeloma), graft-versus-host disease, and many skin disorders (e.g., complication ...
, and the semi-synthesis of
cephalosporin The cephalosporins (sg. ) are a class of β-lactam antibiotics originally derived from the fungus '' Acremonium'', which was previously known as ''Cephalosporium''. Together with cephamycins, they constitute a subgroup of β-lactam antibio ...
antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting pathogenic bacteria, bacterial infections, and antibiotic medications are widely used in the therapy ...
s.Menendez E., Brieva R., Rebolledo F., Gotor V. Optically active (S) ketone and (R) cyanohydrins via an (R)-oxynitrilase catalyzed transformation chemoenzymatic synthesis of 2-cyanotetrahydrofuran and 2-cyanotetrahydropyran. J Chem Soc, Chem Commun. 1995:989-990 The importance of these mandelonitrile synthons makes the HNL class of enzymes a major target for controlled
catalysis Catalysis () is the increase in rate of a chemical reaction due to an added substance known as a catalyst (). Catalysts are not consumed by the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recycles quick ...
that has been optimized through work at the interface of polar and non-polar solvent conditions.


References

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External links

* {{Portal bar, Biology, border=no EC 4.1.2 Flavoproteins Enzymes of known structure