This enzyme belongs to the family of lyases, specifically the aldehyde-lyases, which cleave carbon-carbon bonds. The systematic name 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.
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.[1] Since then, HNLs have been isolated from a wide variety of plants including stone fruits,[2] sorghum grains,[3] millipedes,[4] and passion fruits.[5]
HNLs are peculiar in that, within the same organism and even the same sample, there exist a variety of different isoforms of this enzyme. These isoforms are not able to be determined from one another based on factors influencing activity.[6] This variety also results from macro-heterogeneity, as some isoforms bind FAD 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.[1] Those HNLs that bind FAD do so at a hydrophobic 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 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, whereas HNL I stereo-selectively produce (R)-mandelonitrile.[1]
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.[1] In addition to the study of many isoforms 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 [7] raised antiserum to these specific HNL, which were then applied (with colloidal gold particles in tow) to Black Cherry cotyledon and endosperm. Here it was found that HNL overwhelmingly localizes to the cell walls of these developing plants.[7] It was so enriched in these regions that it was noted upwards of 5% of the cell wall images taken via Electron Microscopy imaged the gold particles that were indirectly labelling these proteins.[7]
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.[5] While Cysteine residues are conserved throughout species in three separate locations (at the N-terminal FAD binding site, and two at the C-terminal 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 grains, 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, HNLs play an essential role in defense of the seed[6][1]
As of late 2007, only one structure has been solved for this class of enzymes, with the PDB accession code 1JU2.
Mechanism of action
Figure 2: The general organic chemistry synthetic pathway for mandelonitrile.
Figure 3: Generalized scheme for the enzymatic cycle and action of HNL. R group indicates a benzene ring.
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 acids 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, whereas it has been shown that HNL activity is highest at a polar-nonpolar interface.[1][13]
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 stereospecificbond formation in (R)-Salbutamol bronchodilators,[14] (S)-amphetamines,[14] (1R, 2S)-(-)-ephedrine bronchodilators,[15] in addition to many others, including Lipitor,[16]Thalidomide,[17] and the semi-synthesis of cephalosporinantibiotics.[18] The importance of these mandelonitrile synthons makes the HNL class of enzymes a major target for controlled catalysis that has been optimized through work at the interface of polar and non-polar solvent conditions.[1][13]
Related Research Articles
In organic chemistry, a cyanohydrin or hydroxynitrile is a functional group found in organic compounds in which a cyano and a hydroxy group are attached to the same carbon atom. The general formula is R2C(OH)CN, where R is H, alkyl, or aryl. Cyanohydrins are industrially important precursors to carboxylic acids and some amino acids. Cyanohydrins can be formed by the cyanohydrin reaction, which involves treating a ketone or an aldehyde with hydrogen cyanide (HCN) in the presence of excess amounts of sodium cyanide (NaCN) as a catalyst:
In biochemistry, flavin adenine dinucleotide (FAD) is a redox-active coenzyme associated with various proteins, which is involved with several enzymatic reactions in metabolism. A flavoprotein is a protein that contains a flavin group, which may be in the form of FAD or flavin mononucleotide (FMN). Many flavoproteins are known: components of the succinate dehydrogenase complex, α-ketoglutarate dehydrogenase, and a component of the pyruvate dehydrogenase complex.
Prunus serotina, commonly called black cherry, wild black cherry, rum cherry, or mountain black cherry, is a deciduous tree or shrub in the rose family Rosaceae. Despite being called black cherry, it is not very closely related to the commonly cultivated cherries, such as sweet cherry, sour cherry and Japanese flowering cherries, which belong to Prunus subg. Cerasus. Instead, P. serotina belongs to Prunus subg. Padus, a subgenus also including Eurasian bird cherry and chokecherry. The species is widespread and common in North America and South America.
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. The flavoproteins are some of the most-studied families of enzymes.
The enzyme argininosuccinate lyase (EC 4.3.2.1, ASL, argininosuccinase; systematic name 2-(N ω-L-arginino)succinate arginine-lyase (fumarate-forming)) catalyzes the reversible breakdown of argininosuccinate:
In enzymology, proline dehydrogenase (PRODH) (EC 1.5.5.2, formerly EC 1.5.99.8) is an enzyme of the oxidoreductase family, active in the oxidation of L-proline to (S)-1-pyrroline-5-carboxylate during proline catabolism. The end product of this reaction is then further oxidized in a (S)-1-pyrroline-5-carboxylate dehydrogenase (P5CDH)-dependent reaction of the proline metabolism, or spent to produce ornithine, a crucial metabolite of ornithine and arginine metabolism. The systematic name of this enzyme class is L-proline:quinone oxidoreductase. Other names in common use include L-proline dehydrogenase, L-proline oxidase,and L-proline:(acceptor) oxidoreductase. It employs one cofactor, FAD, which requires riboflavin (vitamin B2).
Cystathionine beta-lyase, also commonly referred to as CBL or β-cystathionase, is an enzyme that primarily catalyzes the following α,β-elimination reaction
The enzyme phenylalanine ammonia lyase (EC 4.3.1.24) catalyzes the conversion of L-phenylalanine to ammonia and trans-cinnamic acid.:
The enzyme hydroxymandelonitrile lyase catalyzes the chemical reaction
In enzymology, a hydroxynitrilase is an enzyme that catalyzes the chemical reaction
In enzymology, an aminodeoxychorismate synthase is an enzyme that catalyzes the chemical reaction
The enzyme 3α,7α,12α-trihydroxy-5β-cholest-24-enoyl-CoA hydratase (EC 4.2.1.107) catalyzes the chemical reaction
In enzymology, a malate synthase (EC 2.3.3.9) is an enzyme that catalyzes the chemical reaction
In enzymology, a hydroxymandelonitrile glucosyltransferase is an enzyme that catalyzes the chemical reaction
In enzymology, a riboflavin kinase is an enzyme that catalyzes the chemical reaction
In organic chemistry, mandelonitrile is the nitrile of mandelic acid, or the cyanohydrin derivative of benzaldehyde. Small amounts of mandelonitrile occur in the pits of some fruits.
Benzaldehyde (C6H5CHO) is an organic compound consisting of a benzene ring with a formyl substituent. It is among the simplest aromatic aldehydes and one of the most industrially useful.
(R)-prunasin is a cyanogenic glycoside related to amygdalin. Chemically, it is the glucoside of (R)-mandelonitrile.
Aliphatic (R)-hydroxynitrile lyase (EC 4.1.2.46, (R)-HNL, (R)-oxynitrilase, (R)-hydroxynitrile lyase, LuHNL) is an enzyme with systematic name (2R)-2-hydroxy-2-methylbutanenitrile butan-2-one-lyase (cyanide forming). This enzyme catalyses the following chemical reaction:
(S)-hydroxynitrile lyase (EC 4.1.2.47, (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), hydroxynitrilase) is an enzyme with systematic name (S)-cyanohydrin lyase (cyanide forming). This enzyme catalyses the interconversion between cyanohydrins and the carbonyl compounds derived from the cyanohydrin with free cyanide, as in the following two chemical reactions:
References
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↑ Yemm RS, Poulton JE (1986). "Isolation and characterization of multiple forms of mandelonitrile lyase from mature black cherry (Prunus serotina Ehrh.) seeds". Arch. Biochem. Biophys. 247 (2): 440–5. doi:10.1016/0003-9861(86)90604-1. PMID3717954.
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↑ Corson, B. B.; Dodge, R. A.; Harris, S. A.; Yeaw, J. S. (1941). "Mandelic Acid". Organic Syntheses.; Collective Volume, 1, p. 336
↑ Gruber K (2001). "Elucidation of the mode of substrate binding to hydroxynitrile lyase from Hevea brasiliensis". Proteins. 44 (1): 26–31. doi:10.1002/prot.1068. PMID11354003. S2CID19757228.
1 2 Wehtje E, Adlercreutz P, Mattiasson B (1990). "Formation of C-C bonds by mandelonitrile lyase in organic solvents". Biotechnology and Bioengineering. 36 (1): 39–46. doi:10.1002/bit.260360106. PMID18592607. S2CID22377329.
1 2 Effenberger F, Jager J (1997). "Synthesis of the adregenergic bronchodilators (R)-terbutaline and (R)-salbutamol from (R)-cyanohydrins". J. Org. Chem. 62 (12): 3867–3873. doi:10.1021/jo970032d.
↑ Jackson WR., Jacob HA., Matthew BR., Jayatilake GS., Watson KG. Stereoselctive syntheses of ephedrine and related 2-aminoalcohols of high optical purity from protected cyanohydrins. Tetrahedron Lett. 1990; 31:1447-1450
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↑ Ziegler T., Horsch B., Effenberger F., A convenient route to (R)-α-hydroxy carboxylic acids and (2R)-1-amin-2-alkanols from (R)-cyanohydrins. Synthesis 1990:575-578.
↑ 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
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Gross M, Jacobs GH, Poulton JE (1982). "A rapid and sensitive spectrophotometric assay for prunasin hydrolase activity employing purified mandelonitrile lyase". Anal. Biochem. 119 (1): 25–30. doi:10.1016/0003-2697(82)90660-1. PMID6803611.
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Yemm RS, Poulton JE (1986). "Isolation and characterization of multiple forms of mandelonitrile lyase from mature black cherry (Prunus serotina Ehrh.) seeds". Arch. Biochem. Biophys. 247 (2): 440–5. doi:10.1016/0003-9861(86)90604-1. PMID3717954.
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