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

Effenberger, Franz; Hörsch, Brigitte; Förster, Siegfried; Ziegler, Thomas

doi:10.1016/s0040-4039(00)88777-8

Cited by 114 publications

(28 citation statements)

References 19 publications

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“…[31,32] High conversions and ees have been reported for the addition of hydrogen cyanide to 1d but the reaction had to be performed over 6 days [33] (50 U/mmol of HNL from Sorghum bicolor) or at high catalyst loadings to shorten the reaction time [29] (15 min using 1000 U/ mmol of HbHNL). Very good results have also been reported for (S)-2d using mutant strains of MeHNL.…”

Section: M-phenoxybenzaldehydementioning

confidence: 99%

Immobilized Hydroxynitrile Lyases for Enantioselective Synthesis of Cyanohydrins: Sol‐Gels and Cross‐Linked Enzyme Aggregates

Cabirol¹,

Hanefeld²,

Sheldon³

2006

Adv Synth Catal

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The hydroxynitrile lyases (HNLs) from Prunus amygdalus (PaHNL), Manihot esculenta (MeHNL), and Hevea brasiliensis (HbHNL) were successfully immobilized in sol-gels. The cross-linked enzyme aggregate (CLEA) of HbHNL was also prepared. These immobilized enzymes and the commercial PaHNL-and MeHNL-CLEAs were employed for the enantioselective synthesis of cyanohydrins. The sol-gels were highly efficient at low catalyst loading and particularly stable towards the organic solvent (diisopropyl ether) and substrate/product deactivation. The stabilization effect was inconsistent for CLEAs of different HNLs and significant deactivation of PaHNL-and HbHNL-CLEAs in diisopropyl ether was observed. In contrast commercial MeHNL-CLEA proved to be a remarkably robust and efficient biocatalyst in diisopropyl ether.

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Section: M-phenoxybenzaldehydementioning

confidence: 99%

Immobilized Hydroxynitrile Lyases for Enantioselective Synthesis of Cyanohydrins: Sol‐Gels and Cross‐Linked Enzyme Aggregates

Cabirol¹,

Hanefeld²,

Sheldon³

2006

Adv Synth Catal

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“…The FAD content of flavoprotein HNLs is necessary for enzymatic function but the FAD is not involved in a redox reaction [3]. Besides their role in cyanogenesis, HNLs are also of interest for their potential use as a tool for the stereoselective synthesis of cyanohydrins in organic media [11,12]. The HNL from sorghum consists of two A and two B chains comprising a total molecular weight of about 105 000 of which 7 ~o is N-linked carbohydrate.…”

Section: Introductionmentioning

confidence: 99%

Molecular cloning of hydroxynitrile lyase from Sorghum bicolor (L.). Homologies to serine carboxypeptidases

1994

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The heterotetrameric enzyme hydroxynitrile lyase (HNL) from sorghum (EC 4.1.2.11) is involved in the catabolism of the cyanogenic glycoside dhurrin. We have isolated a cDNA clone comprising about 90% of the COOH terminal sequence of a precursor which encodes both subunit of HNL from Sorghum bicolor L. (SbHNL). Hence the subunits of SbHNL must be the result of post-translational processing. The deduced amino acid sequence of HNL shares significant sequence homology with members of the serine carboxypeptidase family. In particular, HNL from sorghum shares the catalytical triad Asp. His, and Ser with these enzymes which evolved in 3 groups of enzymes (carboxypeptidase, chymotrypsin, and subtilisin) by convergent evolution. Moreover, like serine carboxypeptidases, HNL from sorghum consists of two pairs of glycosylated cysteine linked A and B chains forming a heterotetramer of a molecular weight of 105,000 (carboxypeptidases 120,000). Thus, HNL from sorghum closely resembles to serine carboxypeptidases but differs from all other HNLs described so far. Western blotting experiments revealed cross reaction between carboxypeptidase from wheat and anti SbHNL antisera. Therefore, convergent evolution of HNLs from various ancestoral enzymes is conceivable. Hybridization of SbHNL cDNA to northern blots of total RNAs isolated from various organs of young sorghum seedlings shows the same expression pattern of HNL as found by means of western blotting or enzyme assays. Using PCR and Southern blot analysis, we demonstrated that the gene of SbHNL is free of introns. Further sequence analysis of cDNA clones and genomic DNA revealed a stretch of 23 adenine residues in the 3'-untranslated part of the gene. Both, intronless organisation of the gene and a genomic stretch of oligo A suggests that SbHNL may have evolved by a reverse transcription event.

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“…1), are described for several plant species of widely differing phylogenetic origin (1). In recent years these enzymes have received increasing attention due to their potential use as catalysts for the stereoselective synthesis of chiral cyanohydrins (2)(3)(4)(5)(6).…”

mentioning

confidence: 99%

Identification of Potential Active-site Residues in the Hydroxynitrile Lyase from Manihot esculenta by Site-directed Mutagenesis

Wajant

Pfizenmaier

1996

Journal of Biological Chemistry

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The hydroxynitrile lyase from cassava (Manihot esculenta Crantz) (EC 4.1.2.37) catalyzes the decomposition of the achiral ␣-hydroxynitrile acetone cyanohydrin into HCN and acetone during cyanogenesis of damaged plants. This enzyme can also be used for stereoselective synthesis of a wide array of (S)-cyanohydrins by addition of HCN to aldehydes or ketones. Optically active cyanohydrins are interesting intermediates for the synthesis of ␣-hydroxy acids, ␣-hydroxy ketones, or ␤-ethanolamines, all of which are important building blocks in organic synthesis. Inhibition of hydroxynitrile lyase from M. esculenta (MeHNL) by serine-and histidinemodifying reagents suggests involvement of active site seryl and histidyl residues. Furthermore, serine 80 of MeHNL is part of the active site motif Gly-X-Ser-X-Gly/ Ala, often considered as the hallmark of catalytic triads having independently evolved in four groups of enzymes: the ␣/␤ hydrolase fold enzymes, subtilisins, the cysteine proteases, and the eukaryotic serine proteases. By site-directed mutagenesis, three residues critical for enzyme activity have been identified: serine 80, aspartic acid 208, and histidine 236. These residues may be directly involved in MeHNL-catalyzed decomposition of cyanohydrins, providing evidence for a catalytical triad in HNLs, too. The order of the catalytic triad residues in the primary sequence of MeHNL is nucleophilehistidine-acid, suggesting that MeHNL belongs to the ␣/␤ hydrolase fold group of enzymes. In contrast to all other enzymes having a catalytical triad, HNLs catalyze no net hydrolytic reactions.Hydroxynitrile lyases, which catalyze the dissociation of ␣-hydroxynitriles (cyanohydrins) into carbonyle and HCN ( Fig. 1), are described for several plant species of widely differing phylogenetic origin (1). In recent years these enzymes have received increasing attention due to their potential use as catalysts for the stereoselective synthesis of chiral cyanohydrins (2-6).To date, HNLs from various dicotyledons (7, 8) as well as from Sorghum bicolor (SbHNL) 1 (9, 10), a monocotyledone, and Phlebodium aureum (PhaHNL) (11), a fern, have been biochemically characterized. HNLs comprise a heterogenous group of enzymes with homo-and heteromers as the active enzyme, glycoproteins and nonglycoproteins, as well as flavoproteins. The molecular mass of HNL subunits ranges from 20 to 60 kDa (7-11). The heterogeneity of their properties suggests that most of these enzymes have independently evolved (convergent evolution). In fact, the lack of homologies among the amino acid sequences of the recently cloned HNLs from Prunus serotina (PsHNL) (12), Manihot esculenta (Me-HNL) (13), and S. bicolor (14) emphasize this idea. While PsHNL and MeHNL show none or only limited homology to other known proteins (12, 13), SbHNL has astonishingly high sequence homology with serine carboxypeptidases, especially with those from wheat. It appears of particular relevance that SbHNL shares the catalytical triad Ser, Asp, and His with these enzymes (14). The catalytical...

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

Cited by 114 publications

References 19 publications

Immobilized Hydroxynitrile Lyases for Enantioselective Synthesis of Cyanohydrins: Sol‐Gels and Cross‐Linked Enzyme Aggregates

Immobilized Hydroxynitrile Lyases for Enantioselective Synthesis of Cyanohydrins: Sol‐Gels and Cross‐Linked Enzyme Aggregates

Molecular cloning of hydroxynitrile lyase from Sorghum bicolor (L.). Homologies to serine carboxypeptidases

Identification of Potential Active-site Residues in the Hydroxynitrile Lyase from Manihot esculenta by Site-directed Mutagenesis

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