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

Abstract

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 ([RI-mandelonitrile) was purified to apparent homogeneity. The purified holoenzyme is a homomultimer with subunits of M, = 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"C, and a p H optimum at 6.5. In contrast to (RI-MDLs isolated from several species of the Rosaceae family, (R)-MDL from f . dureum 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.

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

“…The above-mentioned similarities between SbHNL and serine carboxypeptidases provoked the question of whether other HNLs also use the catalytical triad motif for catalysis. In support of this idea, almost all HNLs are inhibited by serine/cysteine and/or histidinemodifying agents (11,14,18).In the present study, we have used site-directed mutagenesis to demonstrate that serine 80, which is part of the typical serine protease Gly-X-Ser-X-Gly/Ala consensus motif, is essential for enzyme activity of MeHNL. In addition, site-directed mutagenesis further shows that histidine 236 and aspartic acid 208 are important for enzyme activity.…”

“…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.…”

A recycling pathway for cyanogenic glycosides evidenced by the comparative metabolic profiling in three cyanogenic plant species

“…However, only partially identified compound 17 is apparently a mixture of almost co-eluting vicianin (prunasin-6´-arabinoside) and lucumin (prunasin-6´-xyloside), based on the LC-MS/MS comparison with leaf extracts from Phlebodium aureum and Clerodendrum grayi, which contain high amounts of vicianin and lucumin, respectively [26,27] (Supplementary Figure S3). Fragments at m/z corresponding to mono-and disaccharides Glc, Glc + Glc, and Glc + pentose (as described above) were observed for the respective compounds listed in Table 1B Figure 3.…”

Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese ApricotPrunus mumeand cDNA Cloning and Secretory Expression of One of the Isozymes inPichia pastoris

“…(S)-HNLs are found and investigated in Ximenia americana (XaHNL), 3) Manihot esculenta (MeHNL), 4) Hevea brasiliensis (HbHNL), 5) and Sorghum bicolor (SbHNL). 6) (R)-HNLs are Phlebodium aureum (PhaHNL), 7) Linum usitatissimum (LuHNL), 8,9) Arabidopsis thaliana (AtHNL), 10) and Pr. serotina (PsHNL), [11][12][13] and the most widely investigated HNL is from Pr.…”