Jonathan E. Poulton

Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1.

In plants, Glycoside Hydrolase (GH) Family 1 β-glycosidases are believed to play important roles in many diverse processes including chemical defense against herbivory, lignification, hydrolysis of cell wall-derived oligosaccharides during germination, and control of active phytohormone levels. Completion of the Arabidopsis thalianagenome sequencing project has enabled us, for the first time, to determine the total number of Family 1 members in a higher plant. Reiterative database searches revealed a multigene family of 48 members that includes eight probable pseudogenes. Manual reannotation and analysis of the entire family were undertaken to rectify existing misannotations and identify phylogenetic relationships among family members. Forty-seven members (designated BGLU1 through BGLU47) share a common evolutionary origin and were subdivided into approximately 10 subfamilies based on phylogenetic analysis and consideration of intron–exon organizations. The forty-eighth member of this family (At3g06510; sfr2) is a β-glucosidase-like gene that belongs to a distinct lineage. Information pertaining to expression patterns and potential functions of Arabidopsis GH Family 1 members is presented. To determine the biological function of all family members, we intend to investigate the substrate specificity of each mature hydrolase after its heterologous expression in the Pichia pastoris expression system. To test the validity of this approach, the BGLU44-encoded hydrolase was expressed in P. pastoris and purified to homogeneity. When tested against a wide range of natural and synthetic substrates, this enzyme showed a preference for β-mannosides including 1,4-β-D-mannooligosaccharides, suggesting that it may be involved in A. thaliana in degradation of mannans, galactomannans, or glucogalactomannans. Supporting this notion, BGLU44 shared high sequence identity and similar gene organization with tomato endosperm β-mannosidase and barley seed β-glucosidase/β-mannosidase BGQ60.

Enzymic synthesis of lignin precursors: Purification and properties of the S-adenosyl-l-methionine: Caffeic acid 3-O-methyltransferase from soybean cell suspension cultures

Abstract A 3- O -methyltransferase which catalyzes the methylation of caffeic acid to ferulic acid using S -adenosyl- l -methionine as methyl donor has been isolated and purified about 60-fold from cell suspension cultures of soybean ( Glycine max L., var. Mandarin). The enzyme utilized, in addition to caffeic acid ( K m = 133 μM), 5-hydroxyferulic acid ( K m = 55 μM), 3,4,5-trihydroxy-cinnamic acid ( K m = 100 μM), and protocatechualdehyde ( K m = 50 μM) as substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of ferulic acid, of ortho- , meta- , and para -coumaric acids, and of the flavonoid compounds quercetin and luteolin. The methylation of caffeic acid and 5-hydroxyferulic acid showed a pH optimum at 6.5–7.0. No stimulation of the reaction velocity was observed when Mg 2+ ions were added. EDTA did not inhibit the reaction. The K m for S -adencsyl- l -methionine was 15 μ m . S -Adenosyl- l -homocysteine was a potent competitive inhibitor of S -adenosyl- l -methionine ( K i = 6.9 μM).

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).

UDP-Glucose: Cyanidin 3-O-glucosyltransferase from red cabbage seedlings

Abstract An enzyme, catalysing the glucosylation of cyanidin at the 3-position using uridine diphosphate- D -glucose (UDPG) as glucosyl-donor, has been isolated and purified about 50-fold from young red cabbage ( Brassica oleracea ) seedlings. The pH optimum for this reaction was ca 8 and no additional cofactors were required. The reaction was inhibited by cyanidin (above 0.25 mM) and by very low concentrations of the reaction product cyanidin-3-glucoside (5 μM). The K m values for UDPG and cyanidin were 0.51 and 0.4 mM respectively. In addition to cyanidin the enzyme could also glucosylate the following compounds at the 3-position: pelargonidin, peonidin, malvidin, kaempferol, quercetin, isorhamnetin, myricetin and fisetin. In contrast, cyanidin-3-glucoside, cyanidin-3-sophoroside, cyanidin-3,5-diglucoside, apigenin, luteolin, naringenin and dihydroquercetin were not glucosylated.

Isolation and characterization of multiple forms of mandelonitrile lyase from mature black cherry (Prunus serotina Ehrh.) seeds

Five multiple forms (forms 1-5) of mandelonitrile lyase (EC 4.1.2.10) which catalyze the decomposition of mandelonitrile to benzaldehyde and hydrogen cyanide have been extensively purified from seeds of black cherry (Prunus serotina Ehrh.) by concanavalin A-Sepharose 4B chromatography and chromatofocusing. These forms are monomers which differ only slightly in molecular weight (57,000-59,000) and isoelectric point (4.58-4.63), but heterogeneity in their carbohydrate side-chains was suggested by concanavalin A-Sepharose 4B chromatography. The absorption spectra of the predominating forms 4 and 5 showed maxima of 278, 380, and 460 nm, indicative of flavoprotein character. Detailed comparative kinetic studies of forms 4 and 5 revealed few significant differences in behavior. Both proteins showed pH optima between 6.0 and 7.0, had identical Km values (0.17 mM) for (R,S)-mandelonitrile, and retained similar activities upon storage at 4 and -20 degrees C. Neither form exhibited a metal ion requirement and both were affected similarly by metal salts, beta-mercaptoethanol, and sulfhydryl reagents. Benzoic acid, p-hydroxybenzyl alcohol, and benzyl alcohol inhibited both forms.

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, andPh5) 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.

Tissue Level Compartmentation of (R)-Amygdalin and Amygdalin Hydrolase Prevents Large-Scale Cyanogenesis in Undamaged Prunus Seeds.

Plum (Prunus domestica) seeds, which contain the cyanogenic diglucoside (R)-amygdalin and lesser amounts of the corresponding monoglucoside (R)-prunasin, release the respiratory toxin HCN upon tissue disruption. Amygdalin hydrolase (AH) and prunasin hydrolase (PH), two specific [beta]-glucosidases responsible for hydrolysis of these glucosides, were purified to near homogeneity by concanavalin A-Sepharose 4B and carboxymethyl-cellulose chromatography. Both proteins appear as polypeptides with molecular masses of 60 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but they exhibit different isoelectric points (PH, 5.6–6.0; AH, 7.8–8.2). AH and PH were localized within mature plum seeds by tissue printing, histochemistry, and silver-enhanced immunogold labeling. As was previously shown in black cherry (Prunus serotina) seeds (E.Swain, C.P. Li, J.E. Poulton [1992] Plant Physiol 100: 291–300), AH and PH are restricted to protein bodies of specific procambial cells and are absent from the cotyledonary parenchyma, bundle sheath, and endosperm cells. In contrast, the cyanogenic glycosides in both plum and black cherry seeds, which were detected by tissue printing, occur solely in the cotyledonary parenchyma and are absent from the procambium and endosperm. It is concluded that tissue level compartmentation prevents large-scale cyanoglycoside hydrolysis in intact Prunus seeds.

Two distinct S-adenosyl-l-methionine:3,4-Dihydric phenol 3-O-methyltransferases of phenylpropanoid metabolism in soybean cell suspension cultures

Abstract Two S-adenosyl- l -methionine:3,4-dihydric phenol 3-O-methyltransferases were present in crude extracts from soybean (Glycine max L., var. Mandarin) cell suspension cultures. The enzymes could be clearly distinguished by differences in (a) stability upon storage, (b) elution pattern upon gel chromatography, (c) substrate specificity, and (d) changes in specific activity during the growth cycle of a cell culture. The results from studies of the latter two criteria suggest that one of the methyltransferases, which is specific for substituted cinnamic acids, is related to the formation of lignin, whereas the other is involved in the flavonoid pathway.

Immunocytochemical Localization of Prunasin Hydrolase and Mandelonitrile Lyase in Stems and Leaves of Prunus serotina

In macerates of black cherry (Prunus serotina Ehrh.) leaves and stems, (R)-prunasin is catabolized to HCN, benzaldehyde, and D-glucose by the sequential action of prunasin hydrolase (EC 3.2.1.21) and (R)-(+)-mandelonitrile lyase (EC 4.1.2.10). Immuno-cytochemical techniques have shown that within these organs prunasin hydrolase occurs within the vacuoles of phloem parenchyma cells. In arborescent leaves, mandelonitrile lyase was also located in phloem parenchyma vacuoles, but comparison of serial sections revealed that these two degradative enzymes are usually localized within different cells.

Comparison of kinetic and molecular properties of two forms of amygdalin hydrolase from black cherry (Prunus serotina Ehrh.) seeds.

Two forms of the beta-glucosidase amygdalin hydrolase (AH I and II), which catalyze the hydrolysis of (R)-amygdalin to (R)-prunasin and D-glucose, have been purified over 200-fold from mature black cherry (Prunus serotina Ehrh.) seeds. These proteins showed very similar molecular and kinetic properties but could be resolved by chromatofocusing and isoelectric focusing. AH I and II were monomeric (Mr 60,000) and had isoelectric points of 6.6 and 6.5, respectively. Their glycoprotein character was indicated by positive periodic acid-Schiff staining and by their binding to concanavalin A-Sepharose 4B with subsequent elution by alpha-Me-D-glucoside. Of the natural glycosidic substrates tested, both enzymes showed a pronounced preference for the endogenous cyanogenic disaccharide (R)-amygdalin. They also hydrolyzed at the same active site the synthetic substrates p-nitrophenyl-beta-D-glucoside and 4-methylumbelliferyl-beta-D-glucoside but were inactive towards (R)-prunasin, p-nitrophenyl-alpha-D-glucoside, and 4-methylumbelliferyl-alpha-D-glucoside. Maximum hydrolytic activity was shown in citrate-phosphate buffer in the pH range 4.5-5.0. AH I and II were inhibited competitively by the reaction product (R)-prunasin and noncompetitively (mixed type) by delta-gluconolactone and castanospermine.