Lyases hydroxynitrile

Hydroxynitrile lyases (HNLs) have been found in plants and some insects. In plants their natural function is in the defence against herbivores by release of HCN from hydroxynitriles upon cell damage. The reverse reaction, the formation of (chiral) hydroxynitriles, is interesting for bioorganic chemistry, making FlNLs important biocatalysts for technical applications [14-16]. 

HNLs comprise a heterogenous enzyme family, since hydroxynitrile lyase activity has evolved in different structural frames by convergent evolution [17, 18]. Thus, (S) -specific HNLs based on an a/P-hydrolase fold framework from Manihot esculmta (cassava) [19-21], Hevea hrasilensis (rubber tree) [22-26], and Sorghum hicolor (millet) [27-33] have been described. (R)-specific HNLs based on the structural framework of oxidoreductases were isolated from Linum usitatissimum (flax) [30, 34-37] and Rosaceae (e.g., bitter almonds) [31, 38]. Despite their potential in biocatalysis only few HNLs (from cassava and rubber tree) are available by recombinant gene expression, which is a prerequisite for their technical application [20, 24]. Thus, cloning, recombinant expression, and 

For the synthesis of cyanohydrins nature provides the chemist with R- and S-selective enzymes, the hydroxynitrile lyases (HNL) [4-7]. These HNLs are also known as oxynitrilases and their natural function is to catalyze the release of HCN from natural cyanohydrins like mandelonitrile and acetone cyanohydrin. This is a defense reaction of many plants. It occurs if a predator injures the plant cell. The reaction also takes place when we eat almonds. Ironically the benzaldhyde released together with the HCN from the almonds is actually the flavor that attracts us to eat them. 

Since the release of HCN is a common defense mechanism for plants, the number of available HNLs is large. Depending on the plant family they are isolated from, they can have very different structures some resemble hydrolases or carbox-ypeptidase, while others evolved from oxidoreductases. Although many of the HNLs are not structurally related they all utilize acid-base catalysis. No co-factors need to be added to the reactions nor do any of the HNL metallo-enzymes require metal salts. A further advantage is that many different enzymes are available, R- or S-selective [10]. For virtually every application it is possible to find a stereoselective HNL (Table 5.1). In addition they tend to be stable and can be used in organic solvents or two-phase systems, in particular in emulsions. 

Prunus amygdalus HNL can be employed for the bulk production of (R)-o-chloromandelonitrile, however with a modest enantioselectivity (ee=83%) [9]. When replacing alanine 111 with glycine the mutant HNL showed a remarkably 

Linum usitatissimum HNL, Flax seedlings (R)-butanone cyanohydrin and acetone canohydrin R 

Hevea brasiliensis HNL, Rubber-tree leaves acetone cyanohydrin S 

Their Distribution in Nature, Role and Biochemical Characterisation 

Some 3000 plant species exhibit the ability to release HCN from their tissues, a process which is known as cyanogenesis. This common phenomenon is well recognised in mainly plant sources, as well as a few non-plant sources [6]. 

Presently, the Hnls from eleven cyanogenic plants (from six plant families) have been purified and characterised and the properties of a selection of them are outlined in Table 1. These may be separated into those with (fiavoproteins) 

Plant Part FAD Co-enzyme Molecular Native Weight (kDa) SDS-page Natural substrate (as glycoside) Ref. 

Manihot esculenta (manioc) leaves NO 92-124 28.5-30 acetone cyanohydrin HO CN 33, 34 

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Hydroxynitrile lyase can be used for the decomposition of cyanohydrins with some level of enantioselectivity. " ... 

Griengl reported the first example of hydroxynitrile lyase-catalyzed cyanohydrin formation in a mixed solvent system of [bmim][BF4] and buffer (pH 3.7) (1 1) (Fig. 19). In the reaction, a mixed solvent system was essential, but excellent results were obtained. 

Figure 19 Enantioselective cyanohydrin formation using hydroxynitrile lyase in...

The hydroxynitrile lyase (HNL)-catalyzed addition of HCN to aldehydes is the most important synthesis of non-racemic cyanohydrins. Since now not only (f )-PaHNL from almonds is available in unlimited amounts, but the recombinant (S)-HNLs from cassava (MeHNL) and rubber tree (HbHNL) are also available in giga units, the large-scale productions of non-racemic cyanohydrins have become possible. The synthetic potential of chiral cyanohydrins for the stereoselective preparation of biologically active compounds has been developed during the last 15 years. 

CRYSTAL STRUCTURES OF HYDROXYNITRILE LYASES AND MECHANISM OF CYANOGENESIS... 

Hydroxynitrile lyases (HNLs or oxynitrilases) catalyze C—C bond-forming reactions between an aldehyde or ketone and cyanide to form enantiopure cyanohydrins (Figure 1.15), which are versatile building blocks for the chiral synthesis of amino acids, hydroxy ketones, hydroxy acids, amines and so on [68], Screening of natural sources has led to the discovery of both... 

Purkarthofer, T., Skranc, W., Schuster, C. and Griengl, H. (2007) Potential and capabilities of hydroxynitrile lyases as biocatalysts in the chemical industry. Applied Microbiology and Biotechnology, 76, 309—320. 

Effenberger, F., Forster, S. and Wajant, H. (2000) Hydroxynitrile lyases in stereoselective catalysis. Current... 

Starting from enantiomerically pure 4-methylsulfanyl-mandelonitrile, thiamphenicol and florfenicol have been enantioselectively synthesized (Figure 5.14). The enantiomerically pure 4-methylsulfanyl-mandelonitrile was obtained by hydrocyanation reaction of 4-methy lsulfany 1-benzaldehyde catalyzed by (M)-hydroxynitrile lyase of Badamu (almond from Xinjiang, China) (Prunus communis L. var. dulcis Borkh), which, after an extensive screening, was found to be a highly effective bio-catalyst for this reaction [85]. 

Dreveny, I., Gruber, K., Glieder, A. et al. (2001) The hydroxynitrile lyase from almond a lyase that looks like an oxidoreductase. Structure (London, England 1993), 9, 803-815. 

Dreveny, I., Kratky, C. and Gruber, K. (2002) The active site of hydroxynitrile lyase from Prunus amygdalus modeling studies provide new insights into the mechanism of cyanogenesis. Protein Science A Publication of the Protein Society, 11, 292-300. 

Hasslacher, M., Schall, M., Hayn, M. et al. (1997) High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts. Protein Expression and Purification, 11, 61-71. 

Zuegg, J., Gruber, K., Gugganig, M. et al. (1999) Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis. Protein Science A Publication of the Protein Society, 8, 1990-2000. 

Wagner, U.G., Hasslacher, M., Griengl, H. et al. (1996) Mechanism of cyanogenesis the crystal structure of hydroxynitrile lyase from Hevea brasiliensis. Structure (London, England 1993), 4, 811-822. 

Lauble, H., Miehlich, B., Forster, S. et al. (2002) Crystal structure of hydroxynitrile lyase from Sorghum bicolor in complex with the inhibitor benzoic acid a novel cyanogenic enzyme. Biochemistry, 41, 12043-12050. 

Wajant, H. and Effenberger, F. (1996) Hydroxynitrile lyases of higher plants. The Journal of Biological Chemistry, 377, 611-617. 

Andexer, J., von Langermann, J., Mell, A. et al. (2007) An R-selective hydroxynitrile lyase from Arabidopsis thaliana with an alpha/beta-hydrolase fold. Angewandte Chemie-International Edition, 46, 8679-8681. 

Hernandez, L., Luna, H., Rulz-Teran, F. and Vazquez, A. (2004) Screening for hydroxynitrile lyase activity in crude preparations of some edible plants. Journal of Molecular Catalysis B-Enzymatic, 30, 105-108. 

Forster, S., Roos, J., Effenberger, F. et al. (1996) The first recombinant hydroxynitrile lyase and its application in the synthesis of (S)-cyanohydrins. Angewandte Chemie (International Edition in English), 35, 457 459. 

Glieder, A., Weis, R., Skranc, W. et al. (2003) Comprehensive step-by-step engineering of an (R)-hydroxynitrile lyase for large-scale asymmetric synthesis. Angewandte Chemie-International Edition, 42, 4815 4818. 

Bauer, M., Griengl, H. and Steiner, W. (1999) Kinetic studies on the enzyme (S)-hydroxynitrile lyase from Hevea hyusilh iisis using initial rale methods and progress curve analysis. Biotechnology and Bioengineering, 62,20-29. 

Andexer, J., Guterl, J.-K., Pohl, M. and Eggert, T. (2006) A high-throughput screening assay for hydroxynitrile lyase activity. Chemical Communications, 4201 4203. 

Krammer, B., Rumbold, K., Tschemmernegg, M. et al. (2007) A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening. Journal of Biotechnology, 129, 151-161. 

Willeman, W.F., Straathof, A.J.J. and Heijnen, J.J. (2002) Reaction temperature optimization procedure for the synthesis of (/ )-mandelonitrile by Primus amygdalus hydroxynitrile lyase using a process model approach. Enzyme and Microbial Technology, 30, 200-208. 

Persson, M., Costes, D., Wehtje, E. and Adlercreutz, P. (2002) Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity. Enzyme and Microbial Technology, 30, 916-923. 

Hickel, A., Radke, C.J. and Blanch, H.W. (1999) Hydroxynitrile lyase at the diisopropyl ether/water interface evidence for interfacial enzyme activity. Biotechnology and Bioengineering, 65, 425—136. 

Gaisberger, R.P., Fechter, M.H. and Griengl, H. (2004) The first hydroxynitrile lyase catalysed cyanohydrin formation in ionic liquids. Tetrahedron Asymmetry, 15, 2959-2963. 

Veum, L., Hanefeld, U. and Pierre, A. (2004) The first encapsulation of hydroxynitrile lyase from Hevea brasiliensis in a sol-gel matrix. Tetrahedron, 60, 10419-10425. 

Purkarthofer, T., Gruber, K., Gruber-Khadjawi, M,et al. (2006) A biocatalytic Henry reaction - the hydroxynitrile lyase from Hevea brasiliensis also catalyzes nitroaldol reactions. Angewandte Chemie (International Edition in English), 45, 3454-3456. 

Gruber-Khadjawi, M., Purkarthofer, T. Skranc, W. et al. (2007) Hydroxynitrile lyase-catalyzed enzymatic nitroaldol (Henry) reaction. Advanced Synthesis and Catalysis, 349, 1445-1450. 

Johnson, D.V. and Griengl, H. (1997) The chemoenzymatic synthesis of (5)-13-hydroxyoctadeca-(9Z,ll )-dienoic acid using the hydroxynitrile lyase from Hevea brasiliensis. Tetrahedron, S3, 617-624. 

Rhodococcus erythropolis NCIMB 11540 has been employed as biocatalyst for the conversion of (R)- or (.S )-cyanohydrins to the corresponding (R)- or (S)-a-hydroxycarboxylic acids with an optical purity of up to >99% enatiomeric excess (ee) [27-29] the chiral cyanohydrins can separately be produced using hydroxynitrile lyase from Hevea braziliensis or from Prunus anygdalis [30]. Using the combined NHase-amidase enzyme system of the Rhodococcus erythropolis NCIMB 11 540, the chiral cyanohydrins were first hydrolyzed to the... 

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