Interactions of a fungal thioglucoside glucohydrolase and cruciferous plant epithiospecifier protein to form 1-cyanoepithioalkanes: implications of an allosteric mechanism

Allylglucosinolate is converted to 1-cyano-2,3-epithiopropane by interaction of the thioglucoside glucohydrolase from the fungus Aspergillus sydowi QM 31c and the epithiospecifier protein from Crambe abyssinica. The kinetic evidence presented supports the hypothesis that the epithiospecifier protein interacts with thioglucoside glucohydrolase in an allosteric manner.     

Stereoselectivity of the interactions of thioglucoside glucohydrolase and epithiospecifier protein from various sources

(S)-2-Hydroxy-3-butenylglucosinolate (epi-progoitrin) was converted to erythro- and threo-1-cyamo-2-hydroxy-3,4-epithiobutanes by combinations of thioglucoside glucohydrolase and epithiospecifier protein (ESP) from various sources. In all combinations studied the erythro/threo product ratio was about 1.4, except when the thioglucoside glucohydrolase was from Sinapis alba L. and the ESP from Brassica campestris in which case the ratio was about 1.8.     

Distribution of myrosinase in rapeseed tissues

Immunocytochemical studies on Brassica napus (rapeseed) tissues using a monoclonal antibody against myrosinase (thioglucoside glucohydrolase) showed that the enzyme was only present in a small number of cells. In the developing embryo, scattered myrosinase-containing cells were present in both cotyledons and axis. The enzyme accumulated in these cells during the later stages of seed development, approximately from day 20 until day 40 after pollination. Parallel staining with the immunocytochemical technique and a histochemical method identified these cells as myrosin cells. Myrosinase appeared to be located outside the myrosin grains, although the occasional association with the membrane of the grains also was noted. In leaves, petals, and siliques, scattered parenchyma cells were stained in the mesophyll as well as in the vascular tissue. In young leaves, guard cells also contained myrosinase. The enzyme was also present in xylem cells of the stem.     

The effects of pH on glucosinolate degradation by a thioglucoside glucohydrolase preparation

An active thioglucoside glucohydrolase extract was prepared from commercial mustard powder and its effect on the degradation of two pure glucosinolates was investigated. During reaction in a distilled water medium the pH of the solution decreased markedly and the ratio of products (isothiocyanate and nitrile) varied considerably. After 20–30 min, when the pH had fallen to ca 5.6, isothiocyanate production ceased whilst nitrile continued to be produced and in amounts which increased linearly with time for at least 40 min. This behaviour can be correlated with the changing pH of the medium. In controlled pH experiments it was confirmed that nitrile formation is favoured at lower pH levels and that the ratio of nitrile to isothiocyanate is directly related to the hydrogen ion concentration of the medium. No reason could therefore be found for the observed formation of nitrile in some natural systems at pHs greater than 7.     

Volatile components of cocoa with particular reference to glucosinolate products

The aroma volatiles of raw, fermented and roasted cocoa beans were extracted and concentrated to valid essences using well-established techniques. Analysis by GC and GC/MS showed at least 84 components of which 13 were identified for the first time as cocoa volatiles. In total, ca 5,66 and 65 μg of aroma components were obtained per g of raw, fermented and roasted cocoa beans, respectively. The most abundant groups of volatiles from fermented beans were alcohols (ca40%w/w of the total volatiles) and esters (ca 32%), whilst those from roasted beans were pyrazines (ca 40%) and aldehydes (ca 23%). Trimethyl- and tetramethylpyrazine were also detected in fermented beans, and it is suggested that they contribute to the noticeable cocoa/chocolate aroma of fermented unroasted beans. Phenylacetonitrile, benzyl isothiocyanate and benzyl thiocyanate were all identified amongst cocoa volatiles, together showing the presence of precursor benzylglucosinolate in cocoa. Glucosinolate products were detected in roasted beans, and it seems likely that the enzyme thioglucoside glucohydrolase survived the conditions of roasting. Benzyl thiocyanate was detected only in raw beans, showing that the glucosinolate ‘thiocyanate–forming factor’ did not withstand conditions of fermentation     

Characterization of recombinant nitrile-specifier proteins (NSPs) of Arabidopsis thaliana: Dependency on Fe(II) ions and the effect of glucosinolate substrate and reaction conditions

Glucosinolates are plant secondary metabolites that are part of a plant defence system against pathogens and pests, the myrosinase-glucosinolate system, in which glucosinolates get activated by enzymic degradation through thioglucoside glucohydrolases called myrosinases. Epithiospecifier protein (ESP) and nitrile-specifier proteins (NSPs) divert myrosinase-catalyzed hydrolysis of a given glucosinolate from the formation of isothiocyanate to that of epithionitrile and/or nitrile. As the biological activity of glucosinolate hydrolysis products varies considerably, a detailed characterization of these specifier proteins is of utmost importance to understand their biological role. Therefore, the Arabidopsis thaliana AtNSP1, AtNSP2 and AtNSP5 and a supposed ancestor protein AtNSP-like1 were expressed in Escherichia coli and the activity of the purified recombinant proteins was tested in vitro on three highly different glucosinolates and compared to that of purified AtESP. As previously reported, only AtESP showed epithiospecifier activity on 2-propenylglucosinolate. We further confirmed that purified AtNSP1, AtNSP2 and AtNSP5, but not the ancestor AtNSP-like1 protein, show nitrile-specifier activity on 2-propenylglucosinolate and benzylglucosinolate. We now show for the first time that in vitro AtNSP1, AtNSP2 and AtNSP5 are able to generate nitrile from indol-3-ylmethylglucosinolate. We also tested the effect of different Fe(II) ion concentrations on the nitrile-specifier activity of purified AtNSP1, AtNSP2 and AtNSP5 on 2-propenylglucosinolate and benzylglucosinolate. AtNSP-related nitrile production was highly dependent on the presence of Fe(II) ions in the reaction assay. In the absence of added Fe(II) ions nitriles were only detected when benzylglucosinolate was incubated with AtNSP1. While AtNSP1 also exhibited overall higher nitrile-specifier activity than AtNSP2 and AtNSP5 at a given Fe(II) ion concentration, the pattern of nitrile formation in relation to Fe(II) ion concentrations depended on the AtNSP and the glucosinolate substrate. The pH of the solution also affected the reaction outcome, with a higher proportion of nitrile being produced at the higher pH for AtNSP2 and AtNSP5.     

The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family

A full-length cDNA clone (MB3) and three partial clones (MA1, MB1 and MB2) which encode myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) were isolated from a Sinapis alba (white mustard) cDNA library. Nucleotide sequence analysis of these clones revealed that they are encoded by a gene family. Southern blot analysis with gene-specific probes showed that the gene family consists of a least two subfamilies (MA and MB) each with several members both in S. alba and in Brassica napus (oilseed rape). In Arabidopsis thaliana (wall cress) only three myrosinase genes seem to be present. Northern blot analysis indicated that all the myrosinase mRNA species have the same size, approximately 1.95 kb.     

Cloning,sequencing and analysis of the ggh-A gene encoding a 1,4-β-d-glucan glucohydrolase from Microbispora bispora

The ggh-A gene, encoding a 1,4-β-d-glucan glucohydrolase/β-glucosidase, of Microbispora bispora (Mb) was subcloned and expressed from a 4.0-kb XhoI DNA fragment. The nucleotide sequence of this fragment was determined. Analysis of the sequence revealed one open reading frame (ORF) which encodes a 986-amino-acid (aa) protein with a calculated molecular weight of 107 510. The ggh-A ORF has features typical of an actinomycete gene including high GC content (70.5%) and corresponding biased codon usage. Comparison of the aa sequence of the Mb 1,4-β-d-glucan glucohydrolase (Mbggh-A) with other glycosidases reveals high overall homology to several β-glucosidases and a 1,4-β-d-glucan glucohydrolase belonging to the glycosyl hydrolase family 3. The aa sequence alignments of Mbggh-A and β-glucosidases show that the active site region potentially involves two Asp residues. The aa sequence homology studies revealed a potential two-domain structure for Mbggh-A and other β-glucosidases. Furthermore, Mbggh-A has localized homology to a cellulose-binding domain present in some xylanases. This report is significant, as, to date, 1,4-β-d-glucan glucohydrolases have rarely been reported, though they are assumed to have a critical role in cellulolysis.     

Classic myrosinase‐dependent degradation of indole glucosinolate attenuates fumonisin B1‐induced programmed cell death in Arabidopsis

The mycotoxin fumonisin B1 (FB1) causes the accumulation of reactive oxygen species (ROS) which then leads to programmed cell death (PCD) in Arabidopsis. In the process of studying FB1‐induced biosynthesis of glucosinolates, we found that indole glucosinolate (IGS) is involved in attenuating FB1‐induced PCD. Treatment with FB1 elevates the expression of genes related to the biosynthesis of camalexin and IGS. Mutants deficient in aliphatic glucosinolate (AGS) or camalexin biosynthesis display similar lesions to Col‐0 upon FB1 infiltration; however, the cyp79B2 cyp79B3 double mutant, which lacks induction of both IGS and camalexin, displays more severe lesions. Based on the fact that the classic myrosinase β‐thioglucoside glucohydrolase (TGG)‐deficient double mutant tgg1 tgg2, rather than atypical myrosinase‐deficient mutant pen2‐2, is more sensitive to FB1 than Col‐0, and the elevated expression of TGG1, but not of PEN2, correlates with the decrease in IGS, we conclude that TGG‐dependent IGS hydrolysis is involved in FB1‐induced PCD. Indole‐3‐acetonitrile (IAN) and indole‐3‐carbinol (I3C), the common derivatives of IGS, were used in feeding experiments, and this rescued the severe cell death phenotype, which is associated with reduced accumulation of ROS as well as increased activity of antioxidant enzymes and ROS‐scavenging ability. Despite the involvement of indole‐3‐acetic acid (IAA) in restricting FB1‐induced PCD, feeding of IAN and I3C attenuated FB1‐induced PCD in the IAA receptor mutant tir1‐1 just as in Col‐0. Taken together, our results indicate that TGG‐catalyzed breakdown products of IGS decrease the accumulation of ROS by their antioxidant behavior, and attenuate FB1 induced PCD in an IAA‐independent way.     

The interaction of L-ascorbic acid with the active center of myrosinase.

Only L-ascorbic acid activated plant myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1), whereas ascorbic acid analogs did not. The enzyme protein was conformationally changed by the addition of L-ascorbic acid to the spectrophotometric analysis, approx. 1.5 amino residues appeared on the surface of the enzyme and about 2.3 tryptophan residues were buried in the molecule when 1 mM L-ascorbic acid was added. Optimum temperature for the myrosinase activity was approx. 55 degrees C without L-ascorbic acid, but with L-ascorbic acid it was about 35 degrees C; that for beta-glucosidase activity was the same (55 degrees C) with or without L-ascorbic acid. The effect of chemical modification of the functional groups of myrosinase on the interaction of L-ascorbic acid was investigated and the interaction of L-ascorbic acid with the active center of the enzyme is proposed.     

Hydrolysis of glucosinolates using nylon-immobilized myrosinase to produce pure bioactive molecules

Bioactive compounds were produced from natural glucosinolates, secondary plant metabolites, using myrosinase (thioglucoside glucohydrolase EC 3.2.3.1) isolated from ripe seeds of Sinapis alba. The enzyme was immobilized on granular nylon 6.6 with the crosslinking technique. Immobilized myrosinase displayed extraordinary operational and storage stability. Using a small thermostatted continuous packed-bed bioreactor, the enzyme activity was unchanged after 15 days of continuous use at 37 degrees C and after >1 year of storage at room temperature. The bioreactor was particularly efficient in producing pure isothiocyanates, but it was less efficient for pure nitrile production.     

The Effects of Glucosinolates and Their Breakdown Products on Necrotrophic Fungi

Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.     

Distribution of {beta}-Thioglucosidase Activity in Intact Plants, Cell and Tissue 6Brassica napus L.

Distribution of myrosinase activity in extracts from seeds,intact plants, cell cultures and regenerated callus and plantsof Brassica napus L. was determined by the rate of glucose formationfrom glucosinolate hydrolysis. Calli with shoots and regeneratedplants were obtained from protoplasts or from explants. Of the seedling organs from Brassica napus L. cv. Niklas, hypocotylsshowed the highest myrosinase activity. In cotyledons a nearlyconstant enzyme activity was determined over the first 6 d,followed by a gradual decline. Roots showed a fast decline inenzyme activity over the investigated period. Freshly-isolated protoplasts contained less myrosinase activitythan the original intact tissue. The enzyme activity in developingcalli generally decreased during the first culture periods.After the initial decline a low activity was found which wasstable for a period of more than 2 years. The enzyme activityshowed fluctuations when measured at different times after mediumchange. Protoplast calli with regenerated shoots showed a considerablyhigher myrosinase activity than calli without shoots. Myrosinaseactivity was also found in explant calli including explant callifrom cotyledons and hypocotyls after induction of shoots. Myrosinase activity in seeds from 21 cultivars of Brassica napus,Brassica campestris, Sinapis alba and Raphanus sativus was testedand the highest myrosinase activity was found in seeds fromthe Sinapis alba cultivar Trico while the lowest activity wasfound in the Brassica campestris cultivar Rapido III. Leaf, stem and inflorescence from flowering regenerated or seed-grownplants contained a low but significant myrosinase activity.In contrast, roots showed a high myrosinase activity. The resultsobtained from regenerated plants indicate that the myrosinasesystem is stable in vitro culture, and that the glucosinolate-myrosinasesystem is active in calli tissue. Key words: Myrosinase (thioglucoside glucohydrolase, E.C. 3.2.3.1), in vitro cultures, intact plants     

The myrosinase (thioglucoside glucohydrolase) gene family in Brassicaceae

The glucosinolate hydrolyzing enzymes myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) are encoded by a multigene family consisting of two subgroups. The first two nuclear genes representing each of these two subgroups of the new gene family, Myr1.Bn1 and Myr2.Bn1, from Brassica napus have been cloned and sequenced. Based on conserved regions in cDNA of three species, PCR (polymerase chain reaction) primers were made, and used to amplify and characterize the structure of the myrosinase genes in seven species of Brassiceae. Southern hybridization analysis of PCR products and genomic DNA indicates that myrosinase is encoded by at least 14 genes in B. napus, with similar numbers in the other species of Brassicaceae investigated. The Myr1 gene cloned from B. napus has a 19 amino acid signal peptide and consists of 11 exons of sizes ranging from 54 to 256 bp and 10 introns of sizes from 75 to 229 bp. The Myr2 gene has a 20 amino acid signal peptide and consists of 12 exons ranging in size from 35 to 262 bp and 11 introns of sizes from 81 to 131 bp. The exons from the two genes have 83% homology at the amino acid level. The intron-exon splice sites are of GT..AG consensus type. The signal peptides and presence of sites for N-linked glycosylation, suggest transport and glycosylation through the ER-Golgi complex. The differences between the two genes are discussed on the basis of their predicted expression at different developmental stages in the plant. Both genes show homology to a conserved motif representing the glycosyl hydrolase family of enzymes.     

β-d-Glucosidase: multiplicity of activities and significance to enzymic saccharification of cellulose

Activity measurement of protein components resolved by polyacrylamide gel electrophoresis has indicated that the β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) system is composed of multiple enzymes, each differing in substrate specificity. The proportions of these enzymes varied in preparations obtained from Aspergillus phoenicis, almond emulsion and Trichoderma reesei. The enzyme component showing the highest cellobiase activity was most useful in improving cellulose saccharification by Trichoderma cellulases. The optimum ratio between filter paper and cellobiase activities, expressed in the appropriate units, was 1.0:0.9. The results indicate that for saccharification purposes, the β-d-glucosidase activity should be measured using cellobiose as a substrate, rather than salicin, esculin or 4-nitrophenyl-β-d-glucopyranoside.     

Origin of intestinal disaccharidases of Ascaris suum

Disaccharidases from the gut of Ascaris suum were investigated to determine whether they were synthesized by the worm or whether they were host enzymes adsorbed to the worms' intestinal cells. Alpha-d-glucoside glucohydrolase (maltase) (EC 3.2.1.20), Beta-d-fructofuranoside fructohydrolase (invertase) (EC 3.2.1.26) and 1-glucohydrolase (trehalase) (EC 3.2.1.28) from Ascaris were studied in both a membrane (brush border)-bound and solubilized form with regard to temperature stability and pH optima. Data collected were compared to similar data on hog intestinal enzymes. Worm maltase and trehalase were relatively heat labile, whereas the hog enzymes were more stable to heat inactivation. Worm invertase was heat stable in comparison to the hog enzyme. The pH optima for Ascaris maltase and invertase were different from those of hog disaccharidases, whereas the pH optimum for trehalase from both parasite and host were similar. Tissue homogenates of second-stage larvae contained measurable maltase, but not sucrase, or trehalase activity. Results suggested that Ascaris intestinal disaccharidases represent three distinct enzymes of parasite rather than host origin.     

Rabbit SLC15A1, SLC7A1 and SLC1A1 genes are affected by site of digestion,stage of development and dietary protein content

Peptide transporter 1 (SLC15A1, PepT1), excitatory amino acid transporter 3 (SLC1A1, EAAT3) and cationic amino acid transporter 1 (SLC7A1, CAT1) were identified as genes responsible for the transport of small peptides and amino acids. The tissue expression pattern of rabbit (SLC15A1, SLC7A1 and SLC1A1) across the digestive tract remains unclear. The present study investigated SLC15A1, SLC7A1 and SLC1A1 gene expression patterns across the digestive tract at different stages of development and in response to dietary protein levels. Real time-PCR results indicated that SLC15A1, SLC7A1 and SLC1A1 genes throughout the rabbits’ entire development and were expressed in all tested rabbit digestive sites, including the stomach, duodenum, jejunum, ileum, colon and cecum. Furthermore, SLC7A1 and SLC1A1 mRNA expression occurred in a tissue-specific and time-associated manner, suggesting the distinct transport ability of amino acids in different tissues and at different developmental stages. The most highly expressed levels of all three genes were in the duodenum, ileum and jejunum in all developmental stages. All increased after lactation. With increased dietary protein levels, SLC7A1 mRNA levels in small intestine and SLC1A1 mRNA levels in duodenum and ileum exhibited a significant decreasing trend. Moreover, rabbits fed a normal level of protein had the highest levels of SLC15A1 mRNA in the duodenum and jejunum (P<0.05). In conclusion, gene mRNA differed across sites and with development suggesting time and sites related differences in peptide and amino acid absorption in rabbits. The effects of dietary protein on expression of the three genes were also site specific.     

Myrosinases TGG1 and TGG2 from Arabidopsis thaliana contain exclusively oligomannosidic N-glycans

In all eukaryotes N-glycosylation is the most prevalent protein modification of secretory and membrane proteins. Although the N-glycosylation capacity and the individual steps of the N-glycan processing pathway have been well studied in the model plant Arabidopsis thaliana, little attention has been paid to the characterization of the glycosylation status of individual proteins. We report here the structural analysis of all N-glycans present on the endogenous thioglucoside glucohydrolases (myrosinases) TGG1 and TGG2 from A. thaliana. All nine glycosylation sites of TGG1 and all four glycosylation sites of TGG2 are occupied by oligomannosidic structures with Man₅GlcNAc₂ as the major glycoform. Analysis of the oligomannosidic isomers from wild-type plants and mannose trimming deficient mutants by liquid chromatography with porous graphitic carbon and mass spectrometry revealed that the N-glycans from both myrosinases are processed by Golgi-located α-mannosidases.     

Epithiospecifier protein in turnip and changes in products of autolysis during ontogeny

An epithiospecifier protein present in turnip tissue gives rise to 1-cyano-epithioalkanes during autolysis. Volatile hydrolysis products are produced from glucosinolates during autolysis of seeds, seedlings and plant tissue more than 6 weeks after sowing.