Hydroxynitrile Lyase Research Articles

Induced cyanogenesis from hydroxynitrile lyase and mandelonitrile on wheat with polylactic acid multilayer-coating produces self-defending seeds

Wheat is the predominant crop in the world and vermin infestation remains a serious issue regarding its storage and field cultivation, especially in developing countries. In addition to physical control methods, pesticides are often applied. However, their usage includes a number of concerns regarding environmental and ecological impacts. In the present work an alternative route to protect seeds against herbivore attack is suggested. The seeds were coated with substances capable of cyanogenesis. The precursors were initially in isolated compartments separated through biodegradable polylactide layers. The HCN formation only occurred upon contact of the cyanogenic precursor (mandelonitrile) with a suitable enzyme (hydroxynitrile lyase) and thus needed to be mechanically triggered. This concept is inspired by nature and is based on the protection strategy applied by higher plants, for example apple trees. Further tests showed that the ability for germination was preserved throughout the treatment. Finally, cyanogenesis was followed and quantified in both the liquid and the gas phase and provided HCN in sufficient concentrations to serve as a pest control.

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.

Hydroxynitrile lyases (HNLs) defend plants from herbivores and microbial attack by releasing cyanide from hydroxynitriles. The reverse process has been productively applied to bioorganic syntheses of pharmaceuticals and agrochemicals. To improve our understanding of the catalytic mechanism of HNLs, extensive ab initio QM/MM and classical MM molecular dynamics simulations have been performed to explore the catalytic conversion of cyanohydrins into aldehyde (or ketone) and HCN by hydroxynitrile lyases from Hevea brasiliensis (HbHNLs). It was found that the catalytic reaction approximately follows a two-stage mechanism. The first stage involves two fast processes including the proton abstraction of the substrate through a double-proton transfer and the C-CN bond cleavage, while the second stage concerns HCN formation and is rate-determining. The complete free energy profile exhibits a peak of ∼18 kcal mol(-1). Interestingly, the protonation state of Lys236 influences the efficiency of the enzyme only to some extent, but it changes the entire catalytic mechanism. The dynamical behaviors of substrate delivery and HCN release are basically modulated by the gate movement of Trp128. The remarkable exothermicity of substrate binding and the facile release of HCN may drive the enzyme-catalyzed reaction to proceed along the substrate decomposition efficiently. Computational mutagenesis reveals the key residues which play an important role in the catalytic process.

Uncovering divergent evolution of α/β-hydrolases: a surprising residue substitution needed to convert Hevea brasiliensis hydroxynitrile lyase into an esterase.

Hevea brasiliensis hydroxynitrile lyase (HbHNL) and salicylic acid binding protein 2 (SABP2, an esterase) share 45% amino acid sequence identity, the same protein fold, and even the same catalytic triad of Ser-His-Asp. However, they catalyze different reactions: cleavage of hydroxynitriles and hydrolysis of esters, respectively. To understand how other active site differences in the two enzymes enable the same catalytic triad to catalyze different reactions, we substituted amino acid residues in HbHNL with the corresponding residues from SABP2, expecting hydroxynitrile lyase activity to decrease and esterase activity to increase. Previous mechanistic studies and x-ray crystallography suggested that esterase activity requires removal of an active site lysine and threonine from the hydroxynitrile lyase. The Thr11Gly Lys236Gly substitutions in HbHNL reduced hydroxynitrile lyase activity for cleavage of mandelonitrile 100-fold, but increased esterase activity only threefold to kcat ~ 0.1 min-1 for hydrolysis of p-nitrophenyl acetate. Adding a third substitution - Glu79His - increased esterase activity more than tenfold to kcat ~ 1.6 min-1. The specificity constant (kcat/KM) for this triple substitution variant versus wild type HbHNL shifted more than one million-fold from hydroxynitrile lyase activity (acetone cyanohydrin substrate) to esterase activity (p-nitrophenyl acetate substrate). The contribution of Glu79His to esterase activity was surprising since esterases and lipases contain many different amino acids at this position, including glutamate. Saturation mutagenesis at position 79 showed that 13 of 19 possible amino acid substitutions increased esterase activity, suggesting that removal of glutamate, not addition of histidine, increased esterase activity. Molecular modeling indicates that Glu79 disrupts esterase activity in HbHNL when its negatively charged side chain distorts the orientation of the catalytic histidine. Naturally occurring glutamate at the corresponding location of Candida lipases is uncharged due to other active site differences and does not cause the same distortion. This example of the fine tuning of the same catalytic triad for different types of catalysis by subtle interactions with other active site residues shows how difficult it is to design new catalytic reactions of enzymes.

Mini-Review: Recent Developments in Hydroxynitrile Lyases for Industrial Biotechnology

Hydroxynitrile lyases (HNLs) catalyze the cleavage as well as the formation of cyanohydrins. The latter reaction is valuable for the stereoselective C-C bond formation by condensation of HCN with carbonyl compounds. The resulting cyanohydrins serve as versatile building blocks for a broad range of chemical and enzymatic follow-up reactions. A significant number of (R)- and (S)-selective HNLs are known today and the number is still increasing. HNLs not only exhibit varying substrate scope but also differ in sequence and structure. Tailor-made enzymes for large-scale manufacturing of cyanohydrins with improved yield and enantiomeric excess are very interesting targets, which is reflected in a solid number of patents. This review will complement and extend our recent review with a strong focus on applications of HNLs for the synthesis of highly functionalized, chiral compounds with newest literature, recent and current patent literature.

Chemoenzymatic Asymmetric Total Synthesis of Nonanolide (Z)‐Cytospolides D, E and Their Stereoisomers

AbstractChemoenzymatic asymmetric total synthesis of the (Z)‐isomer of the naturally occurring decanolide cytospolides D, E and six stereoisomers is reported. The main highlight of the synthetic venture involves ring‐closing metathesis (RCM) reaction of a suitably functionalized ester compound, which was assembled by the Yamaguchi coupling of the required acid and alcohol fragments. The alcohol fragment was accessed by two alternative chemoenzymatic processes, one being hydroxynitrile lyase mediated hydrocyanation, whereas lipase‐catalyzed transesterification was the key sep in the second route. The acid fragment was constructed by an enantioselective enzymatic desymmetrization (EED) of prochiral 2‐methyl‐1,3‐propanediol and Corey–Bakshi–Shibata (CBS) mediated stereoselective carbonyl reduction.

HCN production and hydroxynitrile lyase: a natural activity in plants and a renewed biotechnological interest

Over 3,000 plant species are cyanogenic. Cyanogenesis is defined as the hydroxynitrile lyase catalysed release of a cyanide group in the form of HCN and the corresponding aldehyde or ketone. When a plant is attacked, HCN released is a self defence mechanism. A special characteristic of enzymatic reactions is that all enzymatic reactions are reversible-hydroxynitrile lyases can also be used for the synthesis of enantiomerically pure cyanohydrins which are of great importance in industry. This article presents a comprehensive review of the role of hydroxynitrile lyases, both in nature and industry, and an insightful. Areas covered include: history, discovery and natural sources of the hydroxynitrile lyase. Molecular cloning for mass production of this enzyme, including detailed information about several successful recombinant hydroxynitrile lyases is also included.

ChemInform Abstract: A Simple Separation Method for (S)‐Hydroxynitrile Lyase from Cassava and Its Application in Asymmetric Cyanohydrination.

AbstractThe use of a water/iPr2O system with the title enzyme, NaCN and acetic acid is crucial for improving the enantioselectivity by minimizing the non‐enzymatic reaction and randomization.

Biochemical and structural characterization of a novel bacterial manganese‐dependent hydroxynitrile lyase

Hydroxynitrile lyases (HNLs), which catalyse the decomposition of cyanohydrins, are found mainly in plants. In vitro, they are able to catalyse the synthesis of enantiopure cyanohydrins, which are versatile building blocks in the chemical industry. Recently, HNLs have also been discovered in bacteria. Here, we report on the detailed biochemical and structural characterization of a hydroxynitrile lyase from Granulicella tundricola (GtHNL), which was successfully heterologously expressed in Escherichia coli. The crystal structure was solved at a crystallographic resolution of 2.5 Å and exhibits a cupin fold. As GtHNL does not show any sequence or structural similarity to any other HNL and does not contain conserved motifs typical of HNLs, cupins represent a new class of HNLs. GtHNL is metal-dependent, as confirmed by inductively coupled plasma/optical emission spectroscopy, and in the crystal structure, manganese is bound to three histidine and one glutamine residue. GtHNL displayed a specific activity of 1.74 U·mg(-1) at pH 6 with (R)-mandelonitrile, and synthesized (R)-mandelonitrile with 90% enantiomeric excess at 80% conversion using 0.5 m benzaldehyde in a biphasic reaction system with methyl tertiary butyl ether.

ChemInform Abstract: Immobilization of Hydroxynitrile Lyases

AbstractReview: 105 refs.

Chemoenzymatic asymmetric synthesis of fluoxetine, atomoxetine, nisoxetine, and duloxetine

The asymmetric synthesis of two well-known anti-depressant drugs, fluoxetine and duloxetine has been accomplished in a chemoenzymatic manner. The main highlight of the synthesis is the enantioselective cyanohydrin formation by a plant (R)-HNL (hydroxynitrile lyase). The enantiopure cyanohydrins are then synthetically manipulated into the above two drug molecules and two of their structural analogues, atomoxetine and nisoxetine.

Cross linked enzyme aggregates of Prunus armeniaca hydroxynitrile lyase for synthesis of enantiopure (R)-cyanohydrins

Cross linked enzyme aggregates of Prunus armeniaca hydroxynitrile lyase for synthesis of enantiopure (R)-cyanohydrins

Fusion of a Flavin-Based Fluorescent Protein to Hydroxynitrile Lyase from Arabidopsis thaliana Improves Enzyme Stability

Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) was fused to different fluorescent reporter proteins. Whereas all fusion constructs retained enzymatic activity and fluorescence in vivo and in vitro, significant differences in activity and pH stability were observed. In particular, flavin-based fluorescent reporter (FbFP) fusions showed almost 2 orders of magnitude-increased half-lives in the weakly acidic pH range compared to findings for the wild-type enzyme. Analysis of the quaternary structure of the respective FbFP-AtHNL fusion proteins suggested that this increased stability is apparently caused by oligomerization mediated via the FbFP tag. Moreover, the increased stability of the fusion proteins enabled the efficient synthesis of (R)-mandelonitrile in an aqueous-organic two-phase system at a pH of <5. Remarkably, (R)-mandelonitrile synthesis is not possible using wild-type AtHNL under the same conditions due to the inherent instability of this enzyme below pH 5. The fusion strategy presented here reveals a surprising means for the stabilization of enzymes and stresses the importance of a thorough in vitro characterization of in vivo-employed fluorescent fusion proteins.

A simple separation method for (S)-hydroxynitrile lyase from cassava and its application in asymmetric cyanohydrination

Using an acetone precipitation method, crude (S)-hydroxynitrile lyase [(S)-MeHNL] was separated from Munihot esculenta (cassava) leaves, and used directly as biocatalyst to catalyze asymmetric cyanohydrination and produce cyanohydrins with enantiomeric purities (⩾90% ee) significantly greater than those previously reported. The use of a water/i-Pr2O system with an enzyme, NaCN, and appropriate amounts of acetic acid is crucial in improving the stereoselectivity of cyanohydrin formation by minimizing the non-enzymatic reaction and the racemization of the chiral products. The proposed isolation method for crude (S)-MeHNL has a high value because of its simplicity, and low cost as well as the high activity of the crude (S)-MeHNL.

Hydroxynitrile Lyases Do Not Catalyse the Promiscuous Addition of Cyanide to Imines

Hydroxynitrile Lyases Do Not Catalyse the Promiscuous Addition of Cyanide to Imines

Immobilisation of hydroxynitrile lyases

Hydroxynitrile lyases are a versatile group of enzymes that are applied both in the laboratory and on an industrial scale. What makes them particularly interesting is that to date five structurally unrelated categories of hydroxynitrile lyases have been discovered. Given their great importance they have often been immobilised utilising many different methodologies. Therefore the hydroxynitrile lyases are ideally suited to compare different immobilisation methods and their dependence on the structural features of the enzyme in question, since the activity is the same in all cases. This review examines all the different immobilisation methods applied to hydroxynitrile lyases and draws conclusions on the effect of the approach.

Recent Developments in Enzymatic Asymmetric CC Bond Formation

AbstractSome of the recent developments in enzymatic asymmetric CC bond formation are described in this review. The close relationship of biocatalysis and biosynthesis is highlighted with a special emphasis on diversity and biogenesis. One focus of this review is the creation of tetrasubstituted carbon stereocenters. Members of the supposedly well‐known aldolase and hydroxynitrile lyase enzyme families possess the ability to catalyze the formation of tertiary alcohols. In the case of aldolases, this can occur through intramolecular cyclization or intermolecular asymmetric CC bond formation. Thiamine diphosphate‐dependent YerE has been identified as a potent catalyst for the acyloin condensation with ketones as acceptor substrates. C1 transformations such as methylation or carboxylation are catalyzed in an asymmetric manner by enzymes from different classes, for example S‐adenosylmethionine‐dependent (radical) enzymes or NADPH‐dependent oxidoreductases. Insights from biosynthetic and mechanistic studies of enzymatic reactions proceeding via radical intermediates give valuable hints towards possible applications in biocatalysis. Still, the oxygen sensitivity of many of these biocatalysts poses a considerable challenge for practical applications.

Mechanistic and Catalytic Studies of β-Nitroalcohol Crosslinking with Polyamine.

β-Nitroalcohols (βNAs) are promising corneoscleral crosslinking agents for the treatment of diseases such as keratoconus and myopia. Although it is believed that formaldehyde is released from the crosslinking reactions of βNAs, the mechanism by which βNAs react with amine-functionalized polymers has yet to be known. In this study, we present the reaction mechanism of the βNA crosslinking. Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) data provide strong evidence that formaldehyde is released during the reaction. Catalytic studies show that sodium bicarbonate (NaHCO3) and salmon testes DNA accelerate the reaction while hydroxynitrile lyase from Arabidopsis thaliana decelerates the crosslinking reaction. These results suggest that βNAs are potential self-administered crosslinking agents for future clinical use.

Synthesis of Methoxyisopropyl (MIP)-Protected (R)-Mandelonitrile and Derivatives in a Flow Reactor

Cyanohydrins are synthetically versatile chiral building blocks in organic synthesis. They can be conveniently synthesized in enantiomerically pure form via chemoenzymatic hydrogen cyanide addition onto the corresponding aldehyde using hydroxynitrile lyase. Recently, we reported that such transformations can be efficiently carried out in a continuous flow manner using microreactors. Since racemization of enantiopure cyanohydrins occurs readily under slightly basic conditions, they should be protected before the follow-up reactions, preferably under acidic conditions. In this contribution, we demonstrate that the methoxyisopropyl protection of mandelonitrile can be conveniently optimized in an automated microscale continuous flow system and subsequently scaled up under the same conditions by applying a larger flow reactor.

Hydroxynitrile lyases with α/β-hydrolase fold: two enzymes with almost identical 3D structures but opposite enantioselectivities and different reaction mechanisms.

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/β-hydrolase fold, very similar to the homologous, but S-selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser-His-Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)- and (S)-mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)-mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main-chain amide groups and an α-helix dipole very similar to α/β-hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies.

Synthesis of Eight 1‐Deoxynojirimycin Isomers from a Single Chiral Cyanohydrin

AbstractEight configurational 1‐deoxynojirimycin isomers have been synthesized starting from a chiral cyanohydrin as the common precursor. The cyanohydrin chiral pool building block is easily accessible in large quantities by using almond hydroxynitrile lyase as the chiral catalyst in condensing hydrogen cyanide and crotonaldehyde. Our work complements the large body of literature on the synthesis of 1‐deoxynojirimycin derivatives with the distinguishing feature that eight stereoisomers of this important class of glycosidase inhibitors can be derived from a common precursor in an efficient manner.