Beta-peltatin 6-O-methyltransferase from suspension cultures of Linum nodiflorum

S-Adenosyl-L-methionine:beta-peltatin 6-O-methyltransferase was isolated and characterized from cell suspension cultures of Linum nodiflorum L. (Linaceae), a Linum species accumulating aryltetralin lignans such as 6-methoxypodophyllotoxin. The enzyme transfers a methyl group from S-adenosyl-L-methionine to the only free OH-group of beta-peltatin in position 6 thus forming beta-peltatin-A methylether. This reaction is a putative biosynthetic step in the biosynthesis of 6-methoxypodophyllotoxin from deoxypodophyllotoxin. The enzyme has a pH-optimum at pH 7.7 and a temperature optimum at 40 degrees C. The enzyme activity is strongly inhibited by MnSO(4), FeCl(3), FeSO(4) and ZnSO(4) as well as S-adenosyl-homocysteine. Mg(2+) and EDTA did not influence the methylation of beta-peltatin. Substrate saturation curves were obtained for S-adenosyl-methionine and beta-peltatin and apparent K(m)-values of 15 microM and 40 microM, respectively, were determined for these substrates. Substrate inhibition was observed for beta-peltatin. No other lignan substrate tested nor caffeic acid were accepted. The suspension cell line of Linum nodiflorum was characterized with respect to growth, medium alterations and lignan production as well as activity of SAM:beta-peltatin 6-O-methyltransferase. Highest specific activities of beta-peltatin 6-O-methyltransferase were determined on day 7 of the culture period corresponding to the highest levels of 6-methoxypodophyllotoxin on days 7 to 12.     

UDP-glucose:(6-methoxy)podophyllotoxin 7-O-glucosyltransferase from suspension cultures of Linum nodiflorum

Cell cultures of Linum species store 6-methoxypodophyllotoxin (MPTOX), podophyllotoxin (PTOX) and related lignans as O-glucosides. UDP-glucose:(M)PTOX 7-O-glucosyltransferase has been detected and characterised in protein preparations of suspension-cultured cells of Linum nodiflorum L. (Linaceae). The maximal lignan glucoside contents in the cells are preceded by a rapid increase of the specific glucosyltransferase activity on day six of the culture period. MPTOX glucoside is the major lignan with up to 1.18 mg g(-1) of the cell dry wt which is more than 30-fold of the PTOX glucoside content. Of the three aryltetralin lignans tested as substrates, PTOX and MPTOX display comparable apparent K(m) values of 4.7 and 5.4 microM, respectively. 5'-Demethoxy-6-methoxypodophyllotoxin is converted with the highest velocity of 25.2 pkat mg(-1) while also possessing a higher K(m) of 14.7 microM. Two-substrate test series indicate that all three compounds compete for the active site of a single protein. The structurally similar lignan beta-peltatin acts as competitive inhibitor as well. However, the 6-O-glucosidation is most likely catalysed by a separate enzyme. The (M)PTOX 7-O-glucosyltransferase works best at a pH around 9 and a temperature around 35 degrees C. A 15-30% increase of the reaction rate is effected by the addition of 0.9 mM Mn(2+).     

Appearance and immunohistochemical localization of UDP-glucose: coniferyl alcohol glucosyltransferase in spruce (Picea abies (L.) Karst.) seedlings

UDP-glucose: coniferyl alcohol glucosyltransferase was detected in hypocotyls of spruce (Picea abies (L.) Karst.) seedlings. Enzyme activity rose after germination to maximal activity on about the 10th day and then rapidly declined. An antiserum against the glucosyltransferase isolated from cambial sap of spruce (Schmid and Grisebach, 1982, Eur. J. Biochem. 123, 363–370) was employed for localization of the enzyme in cross sections of hypocotyls from 10-d-old spruce seedlings, using the immunofluorescent technique. The results show that the transferase is located predominantly in the epidermal and subepidermal layers and in the vascular bundles. Intracellularly, the enzyme is located in the parietal cytoplasmic layer. The results corroborate the assumption that coniferin (coniferyl alcohol -D-glucoside) participates in lignification of spruce.     

Influence of methyl jasmonate on podophyllotoxin and 6-methoxypodophyllotoxin accumulation in Linum album cell suspension cultures

The accumulation of podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (6MPTOX) was enhanced about twofold in the suspension culture of Linum album line 2-5 aH following the addition of methyl jasmonate (MeJas) to the cultivation medium, reaching 7.69±1.45 mg/g dry weight and 1.11±0.09 mg/g dry weight, respectively. There was no increase in 6MPTOX accumulation following the addition of MeJas to suspension cells of L. album line X4SF, whereas PTOX accumulation was enhanced about tenfold to 0.49±0.10 mg/g dry weight. Phenylalanine ammonia-lyase activity increased immediately after the addition of MeJas to a cell suspension culture of line X4SF, reaching a maximum between 4 h and 1 day after elicitation, while cinnamyl alcohol dehydrogenase activity and the lignin content of the cells were not affected.     

Solubilization and partial characterization of homogalacturonan-methyltransferase from microsomal membranes of suspension-cultured tobacco cells.

The transfer of a methyl group from S-adenosyl-L-methionine onto the carboxyl group of alpha-1,4-linked-galactosyluronic acid residues in the pectic polysaccharide homogalacturonan (HGA) is catalyzed by an enzyme commonly referred to as pectin methyltransferase. A pectin methyltransferase from microsomal membranes of tobacco (Nicotiana tabacum) was previously characterized (F. Goubet, L.N. Council, D. Mohnen [1998] Plant Physiol 116: 337-347) and named HGA methyltransferase (HGA-MT). We report the solubilization of HGA-MT from tobacco membranes. Approximately 22% of the HGA-MT activity in total membranes was solubilized by 0.65% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid containing 1 mM dithioerythritol. The addition of phosphatidylcholine and the methyl acceptors HGA or pectin (30% degree of esterification) to solubilized enzyme increased HGA-MT activity to 35% of total membrane-bound HGA-MT activity. Solubilized HGA-MT has a pH optimum of 7.8, an apparent K(m) for S-adenosyl-L-methionine of 18 microM, and an apparent V(max) of 0. 121 pkat mg(-1) of protein. The apparent K(m) for HGA and for pectin is 0.1 to 0.2 mg mL(-1). Methylated product was solubilized with boiling water and ammonium oxalate, two conditions used to solubilize pectin from the cell wall. The release of 75% to 90% of the radioactivity from the product pellet by mild base treatment showed that the methyl group was incorporated as a methyl ester rather than a methyl ether. The fragmentation of at least 55% to 70% of the radiolabeled product by endopolygalacturonase, and the loss of radioactivity from the product by treatment with pectin methylesterase, demonstrated that the bulk of the methylated product produced by the solubilized enzyme was pectin.     

Justicidin B 7-hydroxylase, a cytochrome P450 monooxygenase from cell cultures of Linum perenne Himmelszelt involved in the biosynthesis of diphyllin

Cell suspension cultures of Linum perenne L. Himmelszelt accumulate justicidin B as the main component together with glycosides of 7-hydroxyjusticidin B (diphyllin). A hypothetical biosynthetic pathway for these compounds is suggested. Justicidin B 7-hydroxylase (JusB7H) catalyzes the last step in the biosynthesis of diphyllin by introducing a hydroxyl group in position 7 of justicidin B. This enzyme was characterized from a microsomal fraction prepared from a Linum perenne Himmelszelt suspension culture for the first time. The hydroxylase activity was strongly inhibited by cytochrome c as well as other cytochrome P450 inhibitors like clotrimazole indicating the involvement of a cytochrome P450-dependent monooxygenase. JusB7H has a pH optimum of 7.4 and a temperature optimum of 26 degrees C. Justicidin B was the only substrate accepted by JusB7H with an apparent K(m) of 3.9+/-1.3 microM. NADPH is predominantly accepted as the electron donor, but NADH was a weak co-substrate. A synergistic effect of NADPH and NADH was not observed. The apparent K(m) for NADPH is 102+/-10 microM.     

Purification and partial amino acid sequences of an esterase from tomato

Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing enzyme activity (0.21-5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of 2.2%. The native enzyme exhibited a M(r) of 26 kDa (gel-filtration chromatography), which was similar to the M(r) determined by SDS-PAGE and MALDI-TOF analysis (M(r) of 28547 kDa). Enzyme kinetics revealed a K(m) value of 15 microM and a V(max) value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 degrees C. The enzyme also efficiently hydrolyzed methyl esters of abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endoproteinase LysC revealed three peptide fragments with 11-14 amino acids. N-Terminal sequencing yielded an additional peptide fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and hydroxynitrile lyases that belong to the alpha/beta hydrolase fold protein superfamily.     

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4′-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8′-linked lignan or 8-5′-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. C. Hano and M. Addi contributed equally to this work.     

Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification

Lignins are phenolic biopolymers synthesized by terrestrial, vascular plants for mechanical support and in response to pathogen attack. Peroxidases have been proposed to catalyse the dehydrogenative polymerization of monolignols into lignins, although no specific isoenzyme has been shown to be involved in lignin biosynthesis. Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA. Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants. Moreover, an Arabidopsis mutant with increased lignin levels compared to wild type shows increased levels of ATP A2 mRNA and of a mRNA encoding an enzyme upstream in the lignin biosynthetic pathway. The substrate specificity of ATP A2 was analysed by X-ray crystallography and docking of lignin precursors. The structure of ATP A2 was solved to 1.45 Å resolution at 100 K. Docking of p-coumaryl, coniferyl and sinapyl alcohol in the substrate binding site of ATP A2 were analysed on the basis of the crystal structure of a horseradish peroxidase C-CN-ferulic acid complex. The analysis indicates that the precursors p-coumaryl and coniferyl alcohols are preferred by ATP A2, while the oxidation of sinapyl alcohol will be sterically hindered in ATP A2 as well as in all other plant peroxidases due to an overlap with the conserved Pro-139. We suggest ATP A2 is involved in a complex regulation of the covalent cross-linking in the plant cell wall.     

Decomposition of 14C-labelled lignin and phenols by a Nocardia sp.

A Gram-positive bacterium which was isolated from a Finnish soil and identified as a Nocardia sp., was able to decompose lignin and to assimilate lignin degradation products as a carbon source. It could release ¹⁴CO₂ from ¹⁴C-labelled methoxyl groups, side chains or ring carbons of coniferyl alcohol dehydropolymers (DHP) and from specifically ¹⁴C-labelled lignin of plant material. Furthermore, it could release ¹⁴CO₂ from phenolcarboxylic and cinnamic acids and alcohols labelled in the OCH₃, COOH groups, side chain or aromatic ring carbons.Non-Common Abbreviations Used DHP dehydropolymers of coniferyl alcohol     

Isolation and characterization of 27-O-demethylrifamycin SV methyltransferase provides new insights into the post-PKS modification steps during the biosynthesis of the antitubercular drug rifamycin B by Amycolatopsis mediterranei S699

The gene rif orf14 in the rifamycin biosynthetic gene cluster of Amycolatopsis mediterranei S699, producer of the antitubercular drug rifamycin B, encodes a protein of 272 amino acids identified as an AdoMet: 27-O-demethylrifamycin SV methyltransferase. Frameshift inactivation of rif orf14 generated a mutant of A. mediterranei S699 that produces no rifamycin B, but accumulates 27-O-demethylrifamycin SV (DMRSV) as the major new metabolite, together with a small quantity of 27-O-demethyl-25-O-desacetylrifamycin SV (DMDARSV). Heterologous expression of rif orf14 in Escherichia coli yielded a 33.8-kDa polyhistidine-tagged polypeptide, which efficiently catalyzes the methylation of DMRSV to rifamycin SV, but not that of DMDARSV or rifamycin W. 27-O-Demethylrifamycin S was methylated poorly, if at all, by the enzyme to produce rifamycin S. The purified enzyme does not require a divalent cation for catalytic activity. While Ca(2+) or Mg(2+) inhibits the enzyme activity slightly, Zn(2+), Ni(2+), and Co(2+) are strongly inhibitory. The K(m) values for DMRSV and S-adenosyl-L-methionine (AdoMet) are 18.0 and 19.3 microM, respectively, and the K(cat) is 87s(-1). The results indicate that DMRSV is a direct precursor of rifamycin SV and that acetylation of the C-25 hydroxyl group must precede the methylation reaction. They also suggest that rifamycin S is not the precursor of rifamycin SV in rifamycin B biosynthesis, but rather an oxidative shunt-product.     

Purification and characterization of malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase (Dp21MaT) from leaves of Digitalis purpurea L

With respect to the cardenolide pathway and the characterization of enzymes involved in the formation of cardenolides, a malonyltransferase, termed malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase (Dp21MaT) has been purified. The enzyme catalyses the transfer of the malonyl moiety from malonyl-coenzyme A to 21-hydroxypregnane substrates. Malonyltransferase activity was checked in several potential starting materials including fresh leaves and cell suspension cultures from different plants. Fresh Digitalis purpurea L. leaves turned out to be the best enzyme source. The purification protocol included ammonium sulphate precipitation, hydrophobic interaction chromatography on Phenylsepharose 6 FF, ion exchange chromatography on Source 30 Q, affinity chromatography on Cibacron Blue 3GA and gel filtration on Superdex 75. Gel filtration and native SDS-PAGE analysis showed that Dp21MaT exists as a monomer with a molecular mass of 27kDa. Its pI, as determined by isoelectric focusing, was 4.66. The enzyme showed maximal activity at pH 6.5 when incubated at 42 degrees C. The energy of activation was 29.28kJmol(-1), whereas that of inactivation was 48.57kJmol(-1). Dp21MaT was purified 252-fold with a yield of about 1%. Hanes plots of kinetic data indicated K(m) values of 99microM (V(max) 47.57microkatkg(-1)) and 28.44microM (V(max) 39.4microkatkg(-1) protein) for 3beta-benzoyloxy-5beta-pregnane-14beta,21-dihydroxy-20-one and malonyl-CoA, respectively.     

Isolation of coniferyl esters from Capsicum baccatum L., and their enzymatic preparation and agonist activity for TRPV1

Coniferyl esters--capsiconiate and dihydrocapsiconiate--were isolated from the fruits of the pepper, Capsicum baccatum L. var. praetermissum. Their structures were determined by spectroscopic methods to be coniferyl (E)-8-methyl-6-nonenoate (capsiconiate) and coniferyl 8-methylnonanoate (dihydrocapsiconiate). This finding was further confirmed by the lipase-catalyzed condensation of coniferyl alcohol with its corresponding fatty acid derivative. The agonist activity of the esters for transient receptor potential vanilloid 1 (TRPV1) was evaluated by conducting an analysis of the intracellular calcium concentrations in TRPV1-expressing HEK293 cells. The EC50 values of capsiconiate and dihydrocapsiconiate were 3.2 and 4.2 microM, respectively.     

Purification and properties of UDP-glucose: coniferyl alcohol glucosyltransferase from suspension culturesof Paul's scarlet rose.

UDP-glucose:coniferyl alcohol glucosyltransferase was isolated from 10-day-old, darkgrown cell suspension cultures of Paul's scarlet rose. The enzyme was purified 120-fold by (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-100. The enzyme has a pH optimum of 7.5 in Tris-HCl buffer, required an -SH group for activity, and is inhibited by ?-chloromercuribenzoate and EDTA. Its molecular weight is estimated to be 52,000. The enzyme is specific for the glucosylation of coniferyl alcohol (K_m 3.3 × 10^?6 M) and sinapyl alcohol (K_m 5.6 × 10^?6 M). With coniferyl alcohol as substrate the apparent K_m value for UDP-glucose is 2 × 10^?6m. The enzyme activity can be detected in a number of callus-tissue and cell-suspension cultures. The role of this enzyme is believed to be to catalyze the transfer of glucose from UDPG to coniferyl (or sinapyl) alcohol as storage intermediates in lignin biosynthesis.     

Aryltetralin-lignan formation in two different cell suspension cultures of Linum album: deoxypodophyllotoxin 6-hydroxylase, a key enzyme for the formation of 6-methoxypodophyllotoxin

Suspension cultures initiated from two different Linum album seedlings accumulate either podophyllotoxin (PTOX, 2.6 mg/g DW) or 6-methoxypodophyllotoxin (6MPTOX, 5.4 mg/g DW) as main lignans. Two molecules of coniferyl alcohol are dimerized to pinoresinol which is converted via several steps into deoxypodophyllotoxin (DOP) which seems to be the branching point to PTOX or 6MPTOX biosynthesis. DOP is hydroxylated at position 7 to give PTOX by deoxypodophyllotoxin 7-hydroxylase (DOP7H). In contrast, 6MPTOX biosynthesis is achieved by DOP hydroxylation at position 6 to beta-peltatin by the cytochrome P450 enzyme deoxypodophyllotoxin 6-hydroxylase (DOP6H). The following methylation to beta-peltatin-A-methylether is catalyzed by beta-peltatin 6-O-methyltransferase (betaP6OMT) from which 6MPTOX is formed by hydroxylation at position 7 by beta-peltatin-A-methylether 7-hydroxylase (PAM7H). DOP6H and betaP6OMT could be characterized in protein extracts from cell cultures of L. flavum and L. nodiflorum, respectively, and here in L. album for the first time. DOP7H and PAM7H activities could not yet be detected with protein extracts. Experiments of feeding DOP together with inhibitors of cytochrome P450 depending as well as dioxygenase enzymes were performed in order to shed light on the type of DOP7H and PAM7H. Growth parameters and specific activities of enzymes from the phenylpropane as well as the lignan specific biosynthetic pathway were measured during a culture period of 16 days. From the enzymes studied only the DOP6H showed a differential activity sustaining the hypothesis that this enzyme is responsible for the differential lignan accumulation in both cell lines.     

Characterization of basic <Emphasis Type="Italic">p</Emphasis>-coumaryl and coniferyl alcohol oxidizing peroxidases from a lignin-forming <Emphasis Type="Italic">Picea abies</Emphasis> suspension culture

A Norway spruce (Picea abies) tissue culture line that produces extracellular lignin into the culture medium has been used as a model system to study the enzymes involved in lignin polymerization. We report here the purification of two highly basic culture medium peroxidases, PAPX4 and PAPX5, and isolation of the corresponding cDNAs. Both isoforms had high affinity to monolignols with apparent K_m values in μM range. PAPX4 favoured coniferyl alcohol with a six-fold higher catalytic efficiency (V_max/K_m) and PAPX5 p-coumaryl alcohol with a two-fold higher catalytic efficiency as compared to the other monolignol. Thus coniferyl and p-coumaryl alcohol could be preferentially oxidized by different peroxidase isoforms in this suspension culture, which may reflect a control mechanism for the incorporation of different monolignols into the cell wall. Dehydrogenation polymers produced by the isoforms were structurally similar. All differed from the released suspension culture lignin and milled wood lignin, in accordance with previous observations on the major effects that e.g. cell wall context, rate of monolignol feeding and other proteins have on polymerisation. Amino acid residues shown to be involved in monolignol binding in the lignification-related Arabidopsis ATPA2 peroxidase were nearly identical in PAPX4 and PAPX5. This similarity extended to other peroxidases involved in lignification, suggesting that a preferential structural organization of the substrate access channel for monolignol oxidation might exist in both angiosperms and gymnosperms.     

Purification and characterization of acetylcoenzyme A: deacetylvindoline 4-O-acetyltransferase from Catharanthus roseus

The enzyme acetylcoenzyme A:deacetylvindoline 4-O-acetyltransferase (EC 2.3.1.-) (DAT), which catalyzes the final step in vindoline biosynthesis in Catharanthus roseus, was purified 3300-fold using ammonium sulfate precipitation followed by gel filtration, anion exchange, hydroxyapatite, and affinity chromatographies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified DAT showed the presence of two major proteins having Mr values of 33,000 and 21,000, whereas native PAGE showed three protein bands, and isoelectric focusing-PAGE one diffuse protein band (pI = 4.7-5.3) plus two minor protein bands (pI = 5.7 and 6.1). Purified DAT possessed Km values of 6.5 microM and 1.3 microM for acetylcoenzyme A and deacetylvindoline, respectively, and Vmax values of 12.6 pkat/microgram protein (acetylcoenzyme A) and 10.1 pkat/micrograms protein (deacetylvindoline). Inhibition of DAT by tabersonine, coenzyme A, and cations (K+, Mg2+, and Mn2+) was observed, while the pH optimum of this enzyme was determined to be 7.5 to 9.     

H(2)O(2) generation during the auto-oxidation of coniferyl alcohol drives the oxidase activity of a highly conserved class III peroxidase involved in lignin biosynthesis

Characterization of lignified Zinnia elegans hypocotyls by both alkaline nitrobenzene oxidation and thioacidolysis reveals that coniferyl alcohol units are mainly found as part of 4-O-linked end groups and aryl-glycerol-beta-aryl ether (beta-O-4) structures. Z. elegans hypocotyls also contain a basic peroxidase (EC 1.11.1.7) capable of oxidizing coniferyl alcohol in the absence of H(2)O(2). Results showed that the oxidase activity of the Z. elegans basic peroxidase is stimulated by superoxide dismutase, and inhibited by catalase and anaerobic conditions. Results also showed that the oxidase activity of this peroxidase is due to an evolutionarily gained optimal adaptation of the enzyme to the microM H(2)O(2) concentrations generated during the auto-oxidation of coniferyl alcohol, the stoichiometry of the chemical reaction (mol coniferyl alcohol auto-oxidized/mol H(2)O(2) formed) being 0.496. These results therefore suggest that the H(2)O(2) generated during the auto-oxidation of coniferyl alcohol is the main factor that drives the unusual oxidase activity of this highly conserved lignin-synthesizing class III peroxidase.     

On the stereoselective synthesis of (+)-pinoresinol in Forsythia suspensa from its achiral precursor, coniferyl alcohol

The residue from Forsythia suspensa stems, upon removal of soluble enzymes, has provided the first evidence for a stereoselective coupling enzyme in lignan biosynthesis. This preparation catalyses the preferred formation (ca 65%) of (+)-[8,8'-14C]pinoresinol from [8-14C]coniferyl alcohol in the absence of exogenously provided cofactors; addition of H2O2 had little effect on enantiomeric composition. However, when NAD and malate were supplied, the stereoselectivity of the coupling reaction was significantly enhanced and pinoresinol consisting of ca 80% of the (+)-antipode was obtained. Clearly, the insoluble residue contains a specific coupling enzyme which catalyses (+)-pinoresinol formation from coniferyl alcohol. By contrast, when [8-14C]sinapyl alcohol was employed as substrate, only racemic syringaresinols were formed: this non-stereoselective peroxidase-catalysed coupling reaction presumably accounts for the low levels of (-)-pinoresinol encountered in this system when coniferyl alcohol is used as a substrate.