Mandelonitrile Lyase Research Articles

Mandelonitrile lyase MDL2-mediated regulation of seed amygdalin and oil accumulation of Prunus Sibirica

BackgroundThe Prunus sibirica seeds with rich oils has great utilization, but contain amygdalin that can be hydrolyzed to release toxic HCN. Thus, how to effectively reduce seed amygdalin content of P. sibirica is an interesting question. Mandelonitrile is known as one key intermediate of amygdalin metabolism, but which mandelonitrile lyase (MDL) family member essential for its dissociation destined to low amygdalin accumulation in P. sibirica seeds still remains enigmatic. An integration of our recent 454 RNA-seq data, amygdalin and mandelonitrile content detection, qRT-PCR analysis and function determination is described as a critical attempt to determine key MDL and to highlight its function in governing mandelonitrile catabolism with low amygdalin accumulation in Prunus sibirica seeds for better developing edible oil and biodiesel in China.ResultsTo identify key MDL and to unravel its function in governing seed mandelonitrile catabolism with low amygdalin accumulation in P. sibirica. Global identification of mandelonitrile catabolism-associated MDLs, integrated with the across-accessions/developing stages association of accumulative amount of amygdalin and mandelonitrile with transcriptional level of MDLs was performed on P. sibirica seeds of 5 accessions to determine crucial MDL2 for seed mandelonitrile catabolism of P. sibirica. MDL2 gene was cloned from the seeds of P. sibirica, and yeast eukaryotic expression revealed an ability of MDL2 to specifically catalyze the dissociation of mandelonitrile with the ideal values of Km (0.22 mM) and Vmax (178.57 U/mg). A combination of overexpression and mutation was conducted in Arabidopsis. Overexpression of PsMDL2 decreased seed mandelonitrile content with an increase of oil accumulation, upregulated transcript of mandelonitrile metabolic enzymes and oil synthesis enzymes (involving FA biosynthesis and TAG assembly), but exhibited an opposite situation in mdl2 mutant, revealing a role of PsMDL2-mediated regulation in seed amygdalin and oil biosynthesis. The PsMDL2 gene has shown as key molecular target for bioengineering high seed oil production with low amygdalin in oilseed plants.ConclusionsThis work presents the first integrated assay of genome-wide identification of mandelonitrile catabolism-related MDLs and the comparative association of transcriptional level of MDLs with accumulative amount of amygdalin and mandelonitrile in the seeds across different germplasms and developmental periods of P. sibirica to determine MDL2 for mandelonitrile dissociation, and an effective combination of PsMDL2 expression and mutation, oil and mandelonitrile content detection and qRT-PCR assay was performed to unravel a mechanism of PsMDL2 for controlling amygdalin and oil production in P. sibirica seeds. These findings could offer new bioengineering strategy for high oil production with low amygdalin in oil plants.

Integrated transcriptomics and metabolomics reveals that regulating biosynthesis and metabolism of HCN and GABA plays a key role in drought resistance of wild soybean

Wild soybeans [Glycine soja (Siebold & Zucc.)] grown in arid areas develop adaptive traits and they are important wild plant resources. In order to reveal the drought-tolerance mechanism in wild soybean, we studied the changes of growth parameters, photosynthetic physiology, and ion physiology of two wild soybean ecotypes under drought stress, and integratedly analyzed the transcriptome and metabolome changes in their leaves. Drought-tolerant wild soybean had greater root/shoot ratio, water use efficiency, and K+ and Ca2+ contents than common wild soybean. In drought-tolerant wild soybean, the gene encoding (R)-mandelonitrile lyase in the pathway of hydrogen cyanide (HCN) biosynthesis and metabolism and the genes encoding serine O-acetyltransferase and aminocyclopropanecarboxylate oxidase in the pathway of 1-aminocyclopropane-1-carboxylate oxidation to produce HCN were down-regulated. In HCN biosynthesis and metabolic pathways of drought-tolerant wild soybean, the content of cysteine as a raw material was significantly reduced, while the content of 3-cyanoalanine as a product was significantly increased. In addition, drought-tolerant wild soybeans up-regulated genes for glutamate synthase and glutamate decarboxylase during synthesis of 4-aminobutyric acid (GABA), accumulating aspartic acid, asparagine, glutamic acid, and GABA; and down-regulated the gene for 4-aminobutyrate-pyruvate transaminase in GABA metabolism. Integrated analysis showed that drought-resistant wild soybean weakened the production of HCN and enhanced the utilization of HCN to avoid HCN toxicity under drought stress. At the same time, it also accumulated GABA to maintain carbon/nitrogen balance and amino acid metabolism. This study provides insights into the genetic mechanism of adaptation to drought by wild soybean, which will be helpful for screening of excellent wild plant resources.

Analysis of cyanide using fluorogenic derivatization and capillary electrophoresis

Samples containing cyanide were incubated at 85 °C in the presence of the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and glutamic acid, and analyzed by capillary electrophoresis utilizing post-separation laser-induced fluorescence detection in a sheath flow cuvette. The separation time on a 25 cm long capillary at 800 Vcm−1 was 3 min with the fluorescent product eluting at 107 s. Flushing of the capillary was not required between runs. Signal was proportional with cyanide concentration from 50 nM to 1.5 μM. LOD and LOQ were determined to be 26 and 87 nM respectively. As an application, free cyanide in five individual apple seeds was measured and found to range from 12 to 86 ng/mg, with a mean of 55 ± 32 ng/mg. As a means for the detection of amygdalin, cyanide was enzymatically produced from amygdalin using the enzymes β-glucosidase and mandelonitrile lyase. The cyanide was then reacted with FQ and injected onto the capillary. Amygdalin was detected at a concentration of 1 μM.

Identification of Pru du 6 as a potential marker allergen for almond allergy.

BackgroundOral food challenges have demonstrated that diagnosis of almond allergy based on extract‐sIgE tests displays low specificity. Molecular allergy diagnosis is expected to improve accuracy, but its value in diagnosing almond allergy remains unknown. The aim of this study was to identify relevant almond allergens and examine their ability to improve almond allergy diagnosis.MethodsIgE‐reactive proteins were purified from almond kernels. IgE binding to almond extract and the allergens was analyzed by quantitative ELISA using sera from 18 subjects with a proven almond allergy. The control group consisted of sera from 18 subjects allergic to peanut and/or tree nuts but tolerant to almond.ResultsThree IgE‐binding proteins were identified: legumin (Pru du 6), alpha‐hairpinin (Pru du 8), and mandelonitrile lyase (Pru du 10). Positive IgE (≥0.35 kU/L) to almond extract showed 94% sensitivity but only 33% specificity. IgE to Pru du 6 maintained high sensitivity (83%) and provided superior specificity (78%). Sera from almond‐allergic subjects had significantly higher IgE levels to almond extract (P < .0001) and Pru du 6 (P < .0001) than sera from tolerant donors. Sensitization to Pru du 6 was highly specific for almond allergy, while frequencies of sensitization to legumins from peanut, walnut, hazelnut, and cashew were similar in both groups. IgE to Pru du 8 and Pru du 10 was less sensitive (41% and 67%), but showed specificities of 100% and 61%.ConclusionThe use of almond allergens markedly increases the diagnostic specificity compared to the extract. Pru du 6 is a potential new molecular marker for almond allergy.

High-throughput in-field bioprospecting for cyanogenic plants and hydroxynitrile lyases

Hydroxynitrile lyases (HNLs) are sought-after, stereo-selective biocatalysts used in the agrochemical, pharmaceutical and fine chemical industries to produce cyanohydrin enantiomers. There are several approaches for the discovery of HNLs, most of which are methodologically demanding and not suitable for high-throughput. Bioprospecting studies to date have also been constrained/limited to commercialised plants or botanical gardens, leaving a vast majority of plant species untested for HNL activity or cyanogenesis. To increase the rate of discovery of HCN liberating plants, we devised a Feigl-Anger microfuge tube that is portable and capable of high throughput detection of naturally cyanogenic plants. A workflow suitable for detecting plant candidates containing extractable, novel HNLs was subsequently applied. In this study, we screened over 600 plants for cyanogenic activity as well as the ability to degrade racemic mandelonitrile. We detected 33 plants able to degrade racemic mandelonitrile, of which, 25 were identified to the species level. Six of these plants were found to be naturally cyanogenic. Protein extracts from 5 of the naturally cyanogenic plants retained the ability to degrade racemic mandelonitrile pointing to five yet undescribed enzymes in the species Achyranthes aspera, Davallia trichomonoides, Morus mesozygia, Polypodium aureum “Mandaianum”, and Thelypteris confluens. In contrast, although Acalypha glabrata was found to be naturally cyanogenic, the protein extract did not break down racemic mandelonitrile. Here, we used racemic mandelonitrile as substrate and detected enzymes with mandelonitrile lyase activity, however, any cyanohydrin could be used as part of the approach taken here to detect novel HNLs specific to the substrate utilised.

Bacillus amyloliquefaciens HG01 induces resistance in loquats against anthracnose rot caused by Colletotrichum acutatum

We aimed to investigate the efficacy of Bacillus amyloliquefaciens HG01 in controlling anthracnose rot caused by Colletotrichum acutatum in postharvest loquats and to explore the probable underlying mechanisms. We observed that relative to the control, HG01 treatment led to lower incidence of disease and smaller diameter of lesions. In HG01-treated loquats, it was observed that there were significant increases in total phenolic and amino acid contents; moreover, HG01-treated fruit appeared to maintain significantly higher sugar and organic acid contents in comparison with the control. Further, the treatment significantly enhanced the activities of two defense-related enzymes, namely chitinase and β-1,3-glucanase. Additionally, after HG01 treatment, the relative expression levels of the following genes were significantly increased: nonexpresser of pathogenesis-related gene, phenylalanine ammonia lyase 2, peroxidase, ethylene-insensitive 3, and mandelonitrile lyase 1. We also noted HG01 significantly inhibited the growth of C. acutatum in vitro, with an inhibition rate of 58.9%. These results suggest that B. amyloliquefaciens HG01 can effectively inhibit anthracnose rot caused by C. acutatum in postharvest loquat fruit, possibly by directly inhibiting growth of the pathogen, and indirectly inducing disease resistance in loquats.

Transcriptome changes specifically associated with apple (Malus domestica) root defense response during Pythium ultimum infection

RNA-seq technology was applied to identify the transcriptomic changes associated with apple root defense response to Pythium ultimum infection. Genes encoding homolog proteins with functions of pathogen detection such as chitin elicitor receptor kinase (CERK) and wall-associated receptor kinase (WAK) were among the differentially expressed apple genes. The biosynthesis and signaling of several plant hormones including ethylene, jasmonate and cytokinin were specifically induced in response to P. ultimum inoculation. Genes encoding enzymes of secondary metabolisms, cell wall fortification and pathogenesis related (PR) protein, laccase, mandelonitrile lyase and cyanogenic beta-glucosidase were consistently up-regulated in the later stages of infection.

Agrobacterium-mediated transformation of black cherry for flowering control and insect resistance

Black cherry is one of the most valuable hardwood species for cabinetry, furniture, and veneer. The goal of this study was to develop transgenic black cherry plants with reproductive sterility and enhanced insect resistance. Black cherry TERMINAL FLOWER 1 (PsTFL1) was overexpressed under the control of the CaMV 35S promoter in black cherry via Agrobacterium-mediated transformation, as a strategy for gene containment. PsTFL1 is a homolog of Arabidopsis TFL1 which is known to play a key role in regulating flowering time by counteracting with FLOWERING LOCUS T and repressing the transcription of the floral-related genes. The elevated expression level of PsTFL1 was proven to be able to significantly delay flowering and cause abnormal floral structure which led to sterility in Arabidopsis. Therefore, the overexpression of PsTFL1 was expected to induce the similar phenotype in black cherry to achieve reproductive sterility. To enhance insect resistance in black cherry, the black cherry endogenous genes encoding prunasin hydrolase isoform 3 (PH3) and mandelonitrile lyase isoform 4 (MDL4) were inserted into black cherry under the control of the phloem-specific promoter rolC or the CaMV 35S promoter. The two enzymes catalyze the hydrolysis of prunasin to mandelonitrile and the dissociation of mandelonitrile to hydrogen cyanide (HCN), respectively, and the overexpression of PH3 or MDL4 might accelerate the release of toxic HCN and lead to an effective protection from cambial-mining insects. Three independent transgenic lines of 35S::PsTFL1, three of rolC::MDL4-FLAG, two of rolC::PH3-FLAG, and eight of 35S::MDL4-FLAG were obtained. The integration of transgenes and the copy number of neomycin phosphotransferase were examined by polymerase chain reaction (PCR) and quantitative PCR (qPCR) analysis. The mRNA levels of PsTFL1, MDL4, and PH3 were examined by real-time qPCR and were compared to the wild-type. The expression level of PsTFL1 in the three 35S::PsTFL1 lines were 3.7–5.8-times higher than that of the wild-type. However, the mRNA level of MDL4 in the 35S::MDL4-FLAG lines and rolC::MDL4-FLAG lines, and the mRNA level of PH3 in the rolC::PH3-FLAG lines showed no significant change indicating that transgene silencing was induced. Western blot analysis was carried out using anti-FLAG antibody to detect the FLAG-tagged PH3 in the transgenic line that had slightly increased mRNA level of PH3, but no signal was detected.

Isolation of Anther-specific Gene Promoters Suitable for Transgene Expression in Rice

To isolate rice anther-specific promoters that allow strong and constitutive expression of transgenes under chilling stress conditions, a genome-wide screen using a microarray was conducted. Six candidate genes for anther-specific expression were selected after comparison of expression levels in leaf and anther tissues from booting stage plants. These genes encoded a mandelonitrile lyase (OsACE1), a sterol desaturase (OsCER1), a MADS-box protein (OsMADS58), a polygalacturonase (OsPGT1), an ERF-like AP2 domain-containing protein (OsERFL1), and a ribosome-inactivating protein-like gene (OsRIP1). Reverse transcriptase-polymerase chain reaction analyses confirmed anther-specific expression of these genes under controlled and in-field chilling conditions. Promoter regions of the selected genes were isolated and fused to the β-glucuronidase (GUS) reporter gene, and the constructs were introduced into rice plants. GUS expression in all transgenic plants except for those carrying OsERFL1and OsRIP1 constructs was only detectable in anthers within the panicles, and no GUS activity was detected in leaves. However, OsERFL1 and OsRIP1 promoters were shown to drive GUS expression in paleae and glumes as well as anthers. GUS expression levels of four transgenic plants were also tested with 2-week-old seedlings and grains. Promoters for OsACE1, OsCER1, OsMADS58, and OsPGT1 were shown to be silent in roots and aerial parts of the seedlings and mature grains. Therefore, these four promoters were deemed suitable for anther-specific transgene expression in rice.

Purification of mandelonitrile lyase from Jiefang loquat seeds

Purification of mandelonitrile lyase from Jiefang loquat seeds

탈지아몬드가루의 Oxynitrilase의 촉매에 의한 Diethyl Malonate의 Aldehyde에 첨가

Defatted almond meal, which contains oxynit-rilase, has been used as a catalyst for the produc-tion of aromatic and aliphatic (R)-cyanohydrins.Oxynitrilase, which is also known as mandeloni-trile lyase (or hydroxynitrile lyase, Hnls), catalyzesthe reversible formation of cyanohydrins from HCNand aldehydes or ketones (Scheme 1).

The apoplastic antioxidant system in Prunus: response to long-term plum pox virus infection

This work describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxymercuribenzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (class I APX) and pHMB-insensitive APX (class III APX), superoxide dismutase (SOD), peroxidase (POX), NADH-POX, and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in class I APX, POX, NADH-POX, and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H2O2 contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H2O2 levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H2O2 levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4-6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. It is concluded that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plants.

Male-fertility genes expressed in male flower buds of Silene latifolia include homologs of anther-specific genes

When the female plant of Silene latifolia is infected with the smut fungus Microbotryum violaceum, its rudimentary stamens develop into anthers which contain fungus teliospores instead of pollen. To identify genes required for maturation of anthers in S. latifolia, we performed a cDNA subtraction approach with healthy male buds and female buds infected with M. violaceum. We isolated five cDNA clones, which were preferentially expressed in healthy male buds during stages associated with a burst in tapetal activity. These five cDNAs are predicted to encode a mandelonitrile lyase protein (SlMDL1), a strictosidine synthase protein (SlSs), a glycosyl hydrolase 17 protein (SlGh17), a proline-rich protein APG precursor (SlAPG), and a chalcone-synthase-like protein (SlChs). All five genes showed expression in both healthy and fungus-infected male buds, but not expressed in either healthy or infected female buds. The first three genes were highly expressed in both tapetum and pollen grains while the last two genes were expressed only inside the tapetum of male flower buds. Phylogenetic analysis results showed that SlChs and SlGh17 belong to anther-specific subgroups of chalcone-synthase-like genes and glycosyl hydrolase 17 family genes, respectively. Our results suggest that the isolated five genes are related to the fertility of the anther leading to the development of fertile pollen. The smut fungus was not able to induce the expression of the five genes in the infected female buds. This raises the possibility that these genes are under the control of master gene(s) on the Y chromosome.

Investigation of the Microheterogeneity and Aglycone Specificity-Conferring Residues of Black Cherry Prunasin Hydrolases

In black cherry (Prunus serotina Ehrh.) seed homogenates, (R)-amygdalin is degraded to HCN, benzaldehyde, and glucose by the sequential action of amygdalin hydrolase (AH), prunasin hydrolase (PH), and mandelonitrile lyase. Leaves are also highly cyanogenic because they possess (R)-prunasin, PH, and mandelonitrile lyase. Taking both enzymological and molecular approaches, we demonstrate here that black cherry PH is encoded by a putative multigene family of at least five members. Their respective cDNAs (designated Ph1, Ph2, Ph3, Ph4, and Ph5) predict isoforms that share 49% to 92% amino acid identity with members of glycoside hydrolase family 1, including their catalytic asparagine-glutamate-proline and isoleucine-threonine-glutamate-asparagine-glycine motifs. Furthermore, consistent with the vacuolar/protein body location and glycoprotein character of these hydrolases, their open reading frames predict N-terminal signal sequences and multiple potential N-glycosylation sites. Genomic sequences corresponding to the open reading frames of these PHs and of the previously isolated AH1 isoform are interrupted at identical positions by 12 introns. Earlier studies established that native AH and PH display strict specificities toward their respective glucosidic substrates. Such behavior was also shown by recombinant AH1, PH2, and PH4 proteins after expression in Pichia pastoris. Three amino acid moieties that may play a role in conferring such aglycone specificities were predicted by structural modeling and comparative sequence analysis and tested by introducing single and multiple mutations into isoform AH1 by site-directed mutagenesis. The double mutant AH ID (Y200I and G394D) hydrolyzed prunasin at approximately 150% of the rate of amygdalin hydrolysis, whereas the other mutations failed to engender PH activity.

An unusual reversible sol-Gel transition phenomenon in organogels and its application for enzyme immobilization in gelatin membranes

An unusual phenomenon is observed for gelatin solutions (1.7-6.8%) in the microemulsion system of 0.3 M bis(2-ethylhexyl)sulfosuccinate sodium salt in isooctane and 14.5% distilled water. Highly viscous gels obtained at temperatures above 30 degreesC become free-flowing liquids at low temperatures (5-10 degreesC). This reversible temperature-dependent sol-gel transition phenomenon is used to immobilize several enzymes, such as lipase from Candida rugosa, alcohol dehydrogenase from baker's yeast, mandelonitrile lyase from Sorghum bicolor, and horseradish peroxidase in the gelatin matrix by solubilizing the enzyme in a microemulsion-based gelatin solution at low temperature (<5 degreesC) and then cross-linking with glutaraldehyde. The enzymes retain 70-80% of their activity after immobilization and can be used in biotransformations in organic solvents without any changes in enantioselectivity. This work provides a unique low-temperature technique for enzyme immobilization in a biocompatible gelatin matrix with a great flexibility of size and shape.

Molecular cloning of the cDNA coding for the (R)-(+)-mandelonitrile lyase of Prunus amygdalus: temporal and spatial expression patterns in flowers and mature seeds.

A gene highly expressed in the floral organs of almond (Prunus amygdalus Batsch), and coding for the cyanogenic enzyme (R)-(+)-mandelonitrile lyase (EC 4.1.2.10), has been identified and the full-length cDNA sequenced. The temporal expression pattern in maturing seeds and during floral development was analyzed by RNA blot, and the highest mRNA levels were detected in floral tissues. The spatial mRNA accumulation pattern in almond flower buds was also analyzed by in-situ hybridization. The mRNA levels were compared during seed maturation and floral development in fruit and floral samples from cultivars classified as homozygous or heterozygous for the sweet-almond trait or homozygous for the bitter trait. No correlation was found between these characteristics and levels of mandelonitrile lyase mRNA, suggesting that the presence of this protein is not the limiting factor in the production of hydrogen cyanide.

Multienzyme Inhibition Biosensor for Amygdalin Measurement

The multienzyme system, containing β′-glucosidase, mandelonitrile lyase and horseradish peroxidase, crosslinked in BSA-gel on the surface of an ion sensitive field effect transistor (ISFET) was used for measuring a cyanogenic glycoside amygdalin which is potentially present in the foods of plant origin. Hydrogen cyanide, produced from amygdalin as a result of β′-glucosidase + mandelonitrile lyase catalyzed reaction, suppressed the activity of horseradish peroxidase, which resulted in a reduction of the biosensor response to hydrogen peroxide. The resulting biosensor can measure amygdalin concentration in the range of 100–300 μM, and can thus be used for the routine amygdalin measurements in food samples where the tolerated concentration of cyanogenic glycosides should not exceed 7 mM.

Analysis of mandelonitrile lyase and beta-glucosidase from sweet almonds by combined electrophoretic techniques.

Almonds are a rich source of mandelonitrile lyase (oxynitrilase) and beta-glucosidase. The isolation of these two enzymes from sweet almonds requires fractional ammonium sulfate precipitation followed by ion-exchange chromatography on diethylaminoethyl-(DEAE) and carboxymethylcellulose (CMC) columns. In the present investigation different electrophoretic techniques such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing in immobilized pH gradients (IEF-IPG), and capillary electrophoresis were used to characterize these two enzymes. For the first time, beta-glucosidase and oxynitrilase were separated in an immobilized pH gradient of one pH unit. Capillary zone electrophoresis (CZE) was an excellent tool for analysis of the purity of enzyme preparations, achieving complete separation of various protein constituents in only 15 min. CZE showed a resolving capacity for the separation of enzyme forms comparable to that of isoelectric focusing in an immobilized pH gradient.

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.

Temporal and spatial expression of amygdalin hydrolase and (R)-(+)-mandelonitrile lyase in black cherry seeds.

In black cherry (Prunus serotina Ehrh.) macerates, the cyanogenic diglucoside (R)-amygdalin undergoes stepwise degradation to HCN catalyzed by amygdalin hydrolase (AH), prunasin hydrolase, and (R)-(+)-mandelonitrile lyase (MDL). A near full-length AH cDNA clone (pAH1), whose insert encodes the isozyme AH I, has been isolated and sequenced. AH I exhibits several features characteristic of beta-glucosidases of the BGA family, including their likely nucleophile center (isoleucine-threonine-glutamic acid-asparagine-glycine) and acid catalyst (asparagine-glutamic acid-proline/isoleucine) motifs. The temporal expression of AH and MDL in ripening fruit was analyzed by northern blotting. Neither mRNA was detectable until approximately 40 days after flowering (DAF), when embryos first became visible to the naked eye. Both mRNAs peaked at approximately 49 DAF before declining to negligible levels when the fruit matured (82 DAF). Taken together with enzyme activity data, these time courses suggest that AH and MDL expression may be under transcriptional control during fruit maturation. In situ hybridization analysis indicated that AH transcripts are restricted to the procambium, whereas MDL transcripts are localized within cotyledonary parenchyma cells. These tissue-specific distributions are consistent with the major locations of AH and MDL protein in mature seeds previously determined by immunocytochemistry (E. Swain, C.P. Li, and J.E. Poulton [1992] Plant Physiol 100:291-300).