Siegfried Förster

Enzyme-catalyzed synthesis of (S)-cyanohydrins and subsequent hydrolysis to (S)-α-hydroxy-carboxylic acids

(S)-Cyanohydrins 2 are obtained with high enantioselectivity from aromatic aldehydes and HCN in the presence of (S)-oxynitrilase (E.C.4.1.2.11). Acid-catalyzed hydrolysis of the cyanohydrins 2 affords the corresponding (S)-α-hydroxy carboxylic acids 3 without racemization.

Hydroxynitrile lyases in stereoselective catalysis

(R)- as well as (S)-cyanohydrins are now easily available as a result of the excellent accessibility, the relatively high stability and the easy handling of hydroxynitrile lyases (HNLs). The optimization of reaction conditions (solvent, temperature, and using site-directed mutagenesis, etc.) has enabled HNL-catalyzed preparations of optically active cyanohydrins on a technical scale. The enantioselectivity of chiral metal-complex-catalyzed additions of trimethylsilyl cyanide to aldehydes has been improved, but is, by far, not yet competitive with the HNL-catalyzed reactions.

Enantioselective hydrolysis of O-acetylmandelonitrile to O-acetylmandelic acid by bacterial nitrilases

Bacteria were enriched from soil samples, using benzylcyanide, α-methyl-, α-ethyl- or α-methoxybenzyl-cyanide as the sole source of nitrogen. All isolated strains belonged to the genus Pseudomonas. Resting cells of the isolates hydrolysed O-acetylmandelonitrile to O-acetylmandelic acid, O-acetylmandelic acid amide and mandelic acid. From racemic O-acetylmandelonitrile all isolates preferentially formed R(−)-acetylmandelic acid ( = d-acetylmandelic acid). The enantioselective hydrolysis of O-acetylmandelonitrile could also be demonstrated in vitro. Crude extracts did not hydrolyse O-acetylmandelic acid amide indicating an enantioselective nitrilase rather than a nitrile hydratase/amidase system.

Substrate Specificity of Mutants of the Hydroxynitrile Lyase from Manihot esculenta

Several tryptophan128‐substituted mutants of the hydroxynitrile lyase from Manihot esculenta (MeHNL) are constructed and applied in the MeHNL‐catalyzed addition of HCN to various aromatic and aliphatic aldehydes as well as to methyl and ethyl ketones to yield the corresponding cyanohydrins. The mutants (especially MeHNL‐W128A) are in most cases superior to the wild‐type (wt) enzyme when diisopropyl ether is used as the solvent. Substitution of tryptophan128 by an alanine residue enlarges the entrance channel to the active site of MeHNL and thus facilitates access of sterically demanding substrates to the active site, as clearly demonstrated for aromatic aldehydes, especially 3‐phenoxybenzaldehyde. These experimental results are in accordance with the X‐ray crystal structure of MeHNL‐W128A. Aliphatic aldehydes, surprisingly, do not demonstrate this reactivity dependence of mutants on substrate bulkiness. Comparative reactions of 3‐phenoxybenzaldehyde with wtMeHNL and MeHNL‐W128A in both aqueous citrate buffer and a two‐phase system of water/methyl tert‐butyl ether again reveal the superiority of the mutant enzyme: 3‐phenoxybenzaldehyde was converted quantitatively into a cyanohydrin nearly independently of the amount of enzyme present, with a space‐time yield of 57 g L−1 h−1.

Purification and Characterization of a Novel (R)-Mandelonitrile Lyase from the Fern Phlebodium aureum

Using high-performance liquid chromatography and nuclear magnetic resonance we identified vicianin as the cyanogenic compound of Phlebodium aureum. The (R)-hydroxynitrile lyase involved during cyanogenesis in the catabolism of the aglycon ([R]-mandelonitrile) was purified to apparent homogeneity. The purified holoenzyme is a homomultimer with subunits of Mr = 20,000. At least three isoforms of the enzyme exist. In contrast to other hydroxynitrile lyases, mandelonitrile lyase (MDL) from P. aureum was not inhibited by sulfhydryl- or hydroxyl-modifying reagents, suggesting a different catalytic mechanism. The enzyme is active over a broad temperature range, with maximum activity between 35 and 50[deg]C, and a pH optimum at 6.5. In contrast to (R)-MDLs isolated from several species of the Rosaceae family, (R)-MDL from P. aureum is not a flavoprotein. The substrate specificity was investigated using immobilized enzyme and diisopropyl ether as solvent. The addition of cyanide to aromatic and heterocyclic carbonyls is catalyzed by this (R)-MDL, whereas aliphatic carbonyls are poorly converted.

Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta

Tryptophan 128 of hydroxynitrile lyase of Manihot esculenta (MeHNL) covers a significant part of a hydrophobic channel that gives access to the active site of the enzyme. This residue was therefore substituted in the mutant MeHNL‐W128A by alanine to study its importance for the substrate specificity of the enzyme. Wild‐type MeHNL and MeHNL‐W128A showed comparable activity on the natural substrate acetone cyanohydrin (53 and 40 U/mg, respectively). However, the specific activities of MeHNL‐W128A for the unnatural substrates mandelonitrile and 4‐hydroxymandelonitrile are increased 9‐fold and ∼450‐fold, respectively, compared with the wild‐type MeHNL. The crystal structure of the MeHNL‐W128A substrate‐free form at 2.1 Å resolution indicates that the W128A substitution has significantly enlarged the active‐site channel entrance, and thereby explains the observed changes in substrate specificity for bulky substrates. Surprisingly, the MeHNL‐W128A–4‐hydroxybenzaldehyde complex structure at 2.1 Å resolution shows the presence of two hydroxybenzaldehyde molecules in a sandwich type arrangement in the active site with an additional hydrogen bridge to the reacting center.

Mechanistic Aspects of Cyanogenesis from Active-Site Mutant Ser80Ala of Hydroxynitrile Lyase from Manihot Esculenta in Complex with Acetone Cyanohydrin.

The structure and function of hydroxynitrile lyase from Manihot esculenta (MeHNL) have been analyzed by X‐ray crystallography and site‐directed mutagenesis. The crystal structure of the MeHNL–S80A mutant enzyme has been refined to an R‐factor of 18.0% against diffraction data to 2.1‐Å resolution. The three‐dimensional structure of the MeHNL–S80A–acetone cyanohydrin complex was determined at 2.2‐Å resolution and refined to an R‐factor of 18.7%. Thr11 and Cys81 involved in substrate binding have been substituted by Ala in site‐directed mutagenesis. The kinetic measurements of these mutant enzymes are presented. Combined with structural data, the results support a mechanism for cyanogenesis in which His236 as a general base abstracts a proton from Ser80, thereby allowing proton transfer from the hydroxyl group of acetone cyanohydrin to Ser80. The His236 imidazolium cation then facilitates the leaving of the nitrile group by proton donating.

Structure of Hydroxynitrile Lyase from Manihot Esculenta in Complex with Substrates Acetone and Chloroacetone: Implications for the Mechanism of Cyanogenesis

The crystal structures of hydroxynitrile lyase from Manihot esculenta (MeHNL) complexed with the native substrate acetone and substrate analogue chloroacetone have been determined and refined at 2.2 A resolution. The substrates are positioned in the active site by hydrogen-bond interactions of the carbonyl O atom with Thr11 OG, Ser80 OG and, to a lesser extent, Cys81 SG. These studies support a mechanism for cyanogenesis as well as for the stereospecific MeHNL-catalyzed formation of (S)-cyanohydrins, which closely resembles the base-catalyzed chemical reaction of HCN with carbonyl compounds.

Acetone cyanohydrin lyase from Manihot esculenta (cassava) is serologically distinct from other hydroxynitrile lyases

Abstract A non-flavoprotein hydroxynitrile lyase clearly different from other hydroxynitrile lyases was isolated from Manihot esculenta (cassava) by combined application of anion exchange chromatography and gel filtration. The purified protein was resolved, upon SDS-PAGE, as a single band of 30 kDa, while the molecular mass of the native enzyme, determined by gel filtration on Superdex 200 was 124 kDa. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited the activity of acetone cyanohydrin lyase, indicating an enzymatically important serine residue. Using immunological techniques, we demonstrate that there is no serological relationship between acetone cyanohydrin layase from cassava and various other hydroxynitrile lyases. This supports the idea that hydroxynitrile lyases have independently evolved from various ancestoral proteins. The hydroxynitrile lyase from cassava is capable of catalyzing the addition of HCN to several aliphatic carbonyls in organic media, demonstrating the potential usefulness of this enzyme in stereoselective synthesis of aliphatic cyanohydrins, which are important building blocks in organic synthesis.

Expression of the Zn2+-containing hydroxynitrile lyase from flax (Linum usitatissimum) in Pichia pastoris - utilization of the recombinant enzyme for enzymatic analysis and site-directed mutagenesis

Abstract Hydroxynitrile lyases (HNL) are involved in the catabolism of cyanogenic glycosides in cyanogenic plants and are powerful tools in the stereoselective synthesis of cyanohydrins. The recent cloning of the hydroxynitrile lyase from flax ( Linum usitatissimum; LuHNL) reveals that this enzyme defines a novel class of HNL. Thorough biochemical and mutational analysis of LuHNL have been hampered by low expression levels of the recombinant enzyme in Escherichia coli . To overcome this impediment, we have cloned a myc-His-tagged LuHNL-cDNA under control of the methanol-inducible AOX1 (alcohol oxidase) promotor of Pichia pastoris and introduced it in the SMD1168 strain. Recombinant LuHNL was kinetically indistinguishable from the authentic flax enzyme. Immobilized LuHNL was used for synthesis of several aliphatic ( R )-cyanohydrins in a preparative scale to analyze the products according to enantiomeric excess and yield of reaction. LuHNL has significant homologies to members of the Zn 2+ -containing alcohol dehydrogenases (Zn 2+ -ADHs). In particular, residues responsible for coordination of Zn 2+ ions or fulfilling structural or functional tasks in Zn 2+ -ADHs are conserved. We found about 2–4 mol zinc per mol of recombinant LuHNL using atom absorption spectroscopy in a non His-tagged version of LuHNL. Using site-directed mutagenesis, we substituted several of the conserved residues against alanine in LuHNL and found that in most cases, HNL-activity was impaired. Hence, it seems that LuHNL and Zn 2+ -ADHs have similar structural requirements with respect to maintaining a catalytically active structure. Residues essentially involved in catalysis of Zn 2+ -ADHs are also of functional importance in LuHNL, suggesting that the removal of the proton from alcohol and cleavage of cyanohydrins can be fulfilled by similar active site structures.