Horseradish peroxidase-catalyzed oligomerization of ferulic acid on a template of a tyrosine-containing tripeptide.

Ferulic acid (FA) is an abundantly present phenolic constituent of plant cell walls. Kinetically controlled incubation of FA and the tripeptide Gly-Tyr-Gly (GYG) with horseradish peroxidase and H2O2 yielded a range of new cross-linked products. Two predominant series of hetero-oligomers of FA linked by dehydrogenation to the peptidyl tyrosine were characterized by electrospray ionization (tandem) mass spectrometry. One series comprises GYG coupled with 4-7 FA moieties linked by dehydrogenation, of which one is decarboxylated. In the second series 4-9 FA moieties linked by dehydrogenation, of which two are decarboxylated, are coupled to the tripeptide. A third series comprises three hetero-oligomers in which the peptidyl tyrosine is linked to 1-3 FA moieties of which none is decarboxylated. Two mechanisms for the formation of the FA-Tyr oligomers that result from the dualistic, concentration-dependent chemistry of FA and their possible role in the regulation of plant cell wall tissue growth are presented.

Plant peroxidases (donor: hydrogen-peroxide oxidoreductases, EC 1.11.1.7) have often been suggested to be involved in the biosynthesis of complex plant cell wall macromolecules constituting lignin and suberin tissues. Lignins are located in the primary and secondary walls of specific plant cells and are synthesized for mechanical strength, defense, and water transport in terrestrial vascular plants. The temporal and spatial correlation between lignin synthesis and peroxidase catalysis has been suggested based on studies of the enzyme, the hydroxycinnamic acid substrates, and the hydrogen peroxide cosubstrate in vivo. In lignin tissues of transgenic plants, the Arabidopsis ATP A2 peroxidase promoter directs GUS reporter gene expression, indicating involvement of the peroxidase in the assembly of the lignin polymer (1). After administration of the reducing substrate [ 13 C]ferulic acid to wheat cell walls for extended duration, elevated levels of ferulic acid in the ligninlike tissues were revealed by in situ solid state 13 C NMR (2). In Pinus taeda cell suspension cultures, lignin synthesis is correlated with the generation of the oxidizing co-substrate H 2 O 2 (3). Suberin provides a physical barrier to moisture loss in plants as well as a defensive shield against pathogens. In the potato, a H 2 O 2 -generating system with NAD(P)H-dependent oxidase-like properties is temporarily associated with suberin synthesis (4), and the polyaromatic domain of suberins in wound-healing potato (Solanum tuberosum) tubers is constituted of polymers derived from hydroxycinnamic acid (5).
Ferulic acid (FA, 1 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid), a hydroxycinnamic acid that is often found to be esterified to plant cell wall polysaccharides (6, 7) is a good substrate for most plant peroxidases. The x-ray structure of horseradish peroxidase C (HRP C) in complex with FA (8) provides molecular insight into the initial abstraction of the hydroxyl hydrogen via one-electron oxidation and subsequent deprotonation of the cation radical, resulting in the FA free radical. Dehydrodimers of FA stemming from radical combination have been identified in plant cell walls (9 -13). In vitro, similar FA dehydrodimers have been identified after incubation of peroxidase with either free FA or FA esterified to pectins and arabinoxylans (14). Recently, it was reported that in incubations with lignin peroxidase, higher polymers of ferulic acid are formed, and the structures of two dehydrotrimers were assigned by 1 H NMR (15).
Tyrosine, both as free amino acid and in peptides or proteins, is also a substrate for many plant peroxidases. After oneelectron oxidation and subsequent deprotonation, a tyrosine radical species is formed that can combine with another tyrosine radical at either the phenolic oxygen or at one of the ortho positions of the aromatic ring (16). Dityrosines and higher oligomers resulting from peroxidase-mediated combination of tyrosine radicals have been characterized for tyrosine-containing peptides (17)(18)(19). The in vivo occurrence of dityrosines in proteins was first demonstrated in insect cuticulum (20). Upon elicitor and wound induction of bean and soybean cell walls, a rapid oxidative cross-linking of the (hydroxy)proline-rich glycoproteins was demonstrated (21). These structural proteins contain highly repetitive peptide sequences, among which the tyrosine-containing Val-Tyr-Lys-Pro-Pro pentapeptide. In vitro, dityrosines stemming from peroxidase catalysis in proteins were demonstrated in soluble collagens and in wheat prolamine (22).
Because both FA and tyrosine are subject to peroxidasemediated oligomerization, we rationalized that hetero-coupling of these substrates should be feasible (19). Such peroxidasemediated hetero-coupling could provide an explanation for the occurrence of protein-carbohydrate complexes in plant cell walls and the incorporation of FA and other hydroxycinnamic derivatives into lignin and suberin tissues on a protein template. From an earlier study with the tripeptide Gly-Tyr-Gly (GYG) and FA, we established conditions that resulted in covalent linkage of FA and the peptidyl tyrosine (19). Evidence was provided for the peroxidase-mediated formation of GYG oligomers linked by dehydrogenation, ranging from dimers to pentamers that are oxidatively cross-linked to either one or two molecules of FA (19). In the present study we have further explored the mechanism of hetero-adduct formation of GYG and FA. Complementary to the aforementioned studies (2,3,5,9,10,13) in which the constituting precursor moieties in plant cell wall tissue were deduced from the in vivo synthesized biopolymer, we now present the in vitro synthesis of the biopolymer by HRP and H 2 O 2 from the hypothesized FA precursor on a tyrosine template.

MATERIALS AND METHODS
Glycine-tyrosine-glycine (GYG) was obtained from Bachem, Bubendorf, Switzerland. HRP (type VI-A) and FA were obtained from Sigma. Hydrogen peroxide (30% v/v) was obtained from Merck. All other chemicals were of analytical grade.
Incubation-Solutions of 12.44 mM ferulic acid and 12.44 mM H 2 O 2 (kept on ice) in 50 mM sodium phosphate, pH 8.0, containing 10% (v/v) acetonitrile were continuously and simultaneously added to the incubation mixture using two peristaltic pumps (LKB Bromma 2232 Microperpex S). The incubation mixture, thermostatted at 20°C, contained 25 mM GYG and 1.25 mg of HRP in an initial volume of 10 ml of 50 mM sodium phosphate, pH 8.0. 20 ml of the FA solution and 20 ml of the H 2 O 2 solution were added over a 15-min period. After another 10 min of incubation, the reaction mixture was frozen in liquid nitrogen, freeze-dried, and stored at 4°C until analysis.
HPLC-MS-For liquid chromatography-MS analysis, 20 l of the reaction mixture (10 mg of lyophilisate redissolved in 100 l of MilliQ water) was separated on a 150 ϫ 2.1-mm Alltima C18 column (Alltech, Breda, The Netherlands) running in 0.03% (v/v) trifluoroacetic acid in water at a flow rate of 0.2 ml/min. Elution was performed with a linear gradient of 0 -40% acetonitrile in 0.03% (v/v) trifluoroacetic acid in water over a 45-min period.
Mass spectrometric analysis was performed with a LCQ ion trap (Finnigan MAT 95, San José, CA) with the use of electrospray ionization and detection in the positive ion mode. The capillary spray voltage was 2.5 kV, and the capillary temperature was 200°C. The instrument was controlled by Xcalibur software. The accuracy of the mass determinations is Ϯ0.5 Da. After a full MS scan, ions with a mass to charge (m/z) ratio within 10 m/z units of the most abundant ion were selected and subjected to collision-induced dissociation (CID, MS 2 ). The five most abundant product ions resulting from the first stage of CID were fragmented further by a second stage of CID (MS 3 ). For nanospray MS, 10 l of the redissolved reaction mixture was diluted with acetonitrile (1:1) and loaded in a conductive metal-coated (coating 3AP) nanospray tip (2-mm internal diameter) (NewObjective, Woburn, MA) using a Gelloader pipette tip (Eppendorf). Nanospray conditions used were 25 units of nitrogen backing pressure and 1.2-kV ionization spray voltage, and the heated desolvation capillary was held at 200°C. 500 scans detected in the positive-ion mode were averaged.

RESULTS
After freeze-drying the incubation mixture, a yellow/orange powder was obtained. Nanoelectrospray MS of the redissolved lyophilisate revealed two dominant series of products ( selection of ions within 10 m/z units from the ion of interest (data not shown). Therefore, the molecular mass of the compounds is considered equal to the apparent m/z values.
The most abundant species is the ion with an apparent m/z value of 829.8 (mono-isotopic). The mass corresponds to a hetero-tetramer of 1 GYG linked by dehydrogenation to a decarboxylated trimer of FA. In the single ion chromatogram of this compound (Fig. 2, panel c), two main isomers, nearly base line-resolved, were detected. Upon collision-induced dissociation of the m/z 829.8 ions, positively charged product ions with m/z 636.0 and 441.8 result (Fig. 3, panel c), stemming from consecutive loss of 2 FA moieties (194 Da each). The fragment ions with m/z 366.8 and 339.0 originate from further dissociation of the m/z 441.8 ion after consecutive loss of Gly and CO from the peptide moiety, resulting in the b2 and a2 ion of the modified tripeptide, as is proved by collision of the isolated m/z 441.8 ion during a second stage of CID (MS 3 ).
The second predominant ion, m/z 1021.9 (mono-isotopic), corresponds to dehydrogenation linkage of another FA moiety to the hetero-tetramer with a m/z of 829.8 and is assigned to a hetero-pentamer of 1 GYG linked by dehydrogenation to 4 FA molecules, of which one is decarboxylated. In the single ion chromatogram of this compound, several co-eluting isomers were detected (Fig. 2, panel d). Upon CID of any of these m/z 1021.9 ions, positively charged product ions with m/z 827.6, 635.4, and 441.8 result (Fig. 3, panel d), stemming from consecutive losses of 3 FA moieties (194,192,and 194 Da). A second stage of CID (MS 3 ) on both the isolated m/z 827.6 and isolated m/z 635.4 fragment ions results in the same m/z 441.8 fragment ion as resulted from CID of the hetero-tetramer, indicating the same structural element in the hetero-pentamer.
In the single ion chromatogram of the third consecutive hetero-oligomer in this series of hetero-oligomers, m/z 1213.8, 1 minor isomer and 2 co-eluting major isomers were detected (Fig. 2, panel e). These are assigned to a hetero-hexamer of 1 GYG peptide linked by dehydrogenation to 5 (Fig. 3, panel e). The three "fingerprint" ions again demonstrate a constituting moiety similar to that in the preceding compounds in this series.
One higher hetero-oligomer in this series was found (Fig. 1 Fig. 2 and therefore starts at c. and this m/z 1407.3 species is assigned to a hetero-heptamer of 1 GYG and 6 FA, of which 1 is decarboxylated. CID of this ionized compound predominantly yielded the fragment ions with m/z 825.9 (loss of 3 FA moieties) and 441.8 (loss of 5 FA moieties) and is concluded to contain the same structural element. The full ion series with m/z values of 829.9, 1021.9, 1214.4, and 1405.8 are assigned to GYG-FA oligomers linked by dehydrogenation containing 3, 4, 5, or 6 FA moieties respectively, each containing the same structural element consisting of a covalent adduct of 1 GYG and decarboxylated FA. The base line-resolved-eluting m/z 341.0 species (Fig. 2, panel b) are assigned to isomeric decarboxylated dehydrodimers of FA.
The third predominant ion in the total ion current, m/z 978.1 (Fig. 2, panel a), is assigned to a hetero-pentamer of 1 GYG linked by dehydrogenation to 4 FA moieties, of which 2 are decarboxylated. Several minor isomers elute separated from two major isomers that co-elute (Fig. 2, panel g). CID of these isomers results in fragment ions with m/z 783.4 and 590.0 (Fig.  3, panel g), corresponding to the consecutive loss of two FA moieties (194 Da). Loss of a third, decarboxylated, FA (148 Da) leads to the main fingerprint fragment ion with m/z 441.8 and the other two fingerprint ions with m/z 366.8 and 339.0 (Fig. 3,  panel g). Therefore the core structural element is concluded to be the same as for the single fold decarboxylated series of hetero-oligomers.
The second predominant product in this ion series, m/z 1170.3 (Fig. 1), corresponds to dehydrogenation linkage of another FA moiety to the 2-fold decarboxylated hetero-pentamer with m/z 978.1 and is assigned to a hetero-hexamer of 1 GYG linked by dehydrogenation to 5 FA molecules, of which 2 are decarboxylated. In the single ion chromatogram of this compound (data not shown), several co-eluting isomers were detected at a retention time similar to that of the m/z 978.1 species. Upon ESI and CID of any of these m/z 1170 species, positively charged ions with m/z 976.8, 783.9, 590.0, and 441.8 result (Fig. 3, panel h), originating from consecutive losses of 4 FA moieties (194, 193, 194, and 148). The full series of ions starting at m/z 978.3 (Fig. 1) are therefore assigned to heterooliogomers of 1 GYG coupled by dehydrogenation with 4 -9 FA moieties, of which 2 are decarboxylated.
The m/z 873.9 ion ( Fig. 1 and the chromatogram in Fig. 2f) is assigned to a hetero-tetramer of 3 nondecarboxylated FA moieties and 1 GYG. This m/z value of this species corresponds to 2 FA molecules linked by dehydrogenation and 1 FA molecule not linked by dehydrogenation to GYG. CID of these ions (Fig.  3, panel f) results in different product ions as compared with the series of 1-and 2-fold decarboxylated hetero-oligomers, and the fingerprint ions from the former series are completely absent. This implies that the ion with a m/z of 873.9 cannot contain the m/z 441.9 moiety like the former series of heterooligomers. The main fragment ion is that with m/z 295.8, corresponding to the GYG ion after loss of the FA moieties. No higher hetero-oligomers containing exclusively nondecarboxylated FA moieties were found. However, two smaller nondecarboxylated hetero-oligomers, m/z 488 (hetero-dimer) and m/z 680 (hetero-trimer), were formed in minor amounts (Fig. 1). Interestingly, these compounds were main products in a previous incubation (19). DISCUSSION In this study it is shown that the kinetically controlled incubation of FA and GYG with peroxidase and H 2 O 2 results in a complex range of (isomeric) products of different degrees of polymerization. Because isolation of these products for NMR characterization is not straightforward, mass spectrometry including a first and second stage of collision-induced dissociation was the method of choice to elucidate the range of products and their compositional structural elements.
In both the series of 1-fold decarboxylated and the series of 2-fold decarboxylated hetero-oligomers the difference in mass between the compounds was always 192 Da, a mass difference expected for dehydrogenation linkage of FA to each preceding compound in a constitutive fashion. Unambiguous assignment of the ions of the compounds to the constituting moieties of the two series of hetero-oligomers is provided by CID. Upon CID of a hetero-oligomer, the covalent linkages in the proximity of the C-C bond that connects the four aromatic moieties A, B, C, and Tyr (Fig. 4, structure 5h e.g.) are most susceptible to fragmentation due to the high probability of the presence of the proton in this acetal-type structural moiety. After loss of two neutral FA moieties, the 441.9 fragment results (Fig. 4, structure 5h 4 ). Further fragmentation of this fragment (MS 3 ) resulted in the b2 and a2 ions (Fig. 4, structures 5h 6 and 5h 7 ) that further validates the assignment of the 441.9 fingerprint ion as GYG linked to the decarboxylated FA moiety. The presence of the fragment ion of 441.9 (Fig. 3, panels c, d, e, g, and h) in all MS 2 spectra of compounds of the one-or 2-fold decarboxylated hetero-oligomers and the fragments of 367 and 339 in the MS 3 spectrum of the parent mass 441.9 clearly indicates that the GYG peptide bound to the decarboxylated FA moiety (Fig. 4, all structures, ring C) is always one of the constituting moieties of at least 1 isomer of the 1-and 2-fold decarboxylated heterooligomers. Most of the other, isomeric hetero-tetramers (Fig.  6b, structures 5a-h) are unlikely to lead to the fingerprint fragment ions as outlined for isomer 5h in Fig. 4. However, these species may well contribute to the total ion current of the compound of interest but not participate in the fragmentation pattern of a co-eluting isomer.
In contrast to the 1-and 2-fold decarboxylated hetero-oligomers, the fragmentation of the 873.9 species (Fig. 3, panel f) does not lead to product ions corresponding to the m/z 441.9 structural element (Fig. 4, structure 5h 4 ). This nondecarboxylated hetero-oligomer results mainly in an m/z 295.8 product ion (Fig. 3, panel f), which corresponds to GYG with no decarboxylated FA moiety attached. This distinct product ion pattern was also found for nondecarboxylated FA-GYG-FA and FA-GYG in a previous study, where GYG was kept in excess over FA (19) during the incubation. Higher hetero-oligomers of this type were not found in those studies nor in the present one.
This discrepancy between the wide range of decarboxylated, further oligomerized hetero-oligomers and the limited range of nondecarboxylated, maximally tetrameric hetero-oligomers suggests two different mechanisms for the covalent attachment of FA to the tyrosine template. Under the conditions applied, the main route occurs via radical attack on the vinylic bond of the decarboxylated FA dimer (Fig. 5a, structures 2a and 2b), whereas the minor route proceeds via de-or nondehydrogenation addition of the non-decarboxylated monomeric FA radical. The main route deduced from the present data is supported by a recently proposed mechanism for the lignin peroxidase-catalyzed homo-trimerization of FA (15). This mechanism comprises an attack by a phenoxy-FA radical (Fig. 5a, structure 1a) on the vinylic bond of a decarboxylated FA dehydrodimer (Fig.  5, structures 2a and 2b). The initially formed trimeric FA radical species resulting from this attack (Fig. 5b, structures 3a-d) can be oxidized and, after deprotonation, result in a FA trimer, as was authenticated by one-and two-dimensional 1 H NMR (15). Decarboxylated dehydrodimers of FA were also formed in the present HRP-mediated incubation of GYG with FA (Fig. 2,  panel b).
Because GYG radicals are continuously present (Fig. 6a,  structures 4a and 4b) in the hetero-incubation, as follows from the presence of GYG dimers (data not shown), two routes can directly lead to a hetero-oligomer. Combination of the trimeric FA radical (Fig. 5b, structures 3a-d) with the peptidyl tyrosine radical of an oxidized GYG (Fig. 5a, structures 4a or 4b) results in a hetero-tetramer comprising 3 FA and 1 GYG (Fig. 6b,  structures 5a-h). Attack of the GYG radical on the vinylic bond of the decarboxylated FA dimer and subsequent combination of the resulting hetero-trimeric radical with another FA radical also leads to the hetero-tetramer, whereas attack of the heterotrimeric radical on another decarboxylated FA dimer leads to a 2-fold decarboxylated hetero-pentamer. These routes, both via decarboxylated FA dimers, appear to be important in the oligomerization to higher FA oligomers on the peptide since the higher hetero-oligomers always are 1-or 2-fold-decarboxylated. Any of the 1-fold decarboxylated hetero-tetramers (Fig. 6b, structures 5a-h) or 2-fold decarboxylated hetero-pentamers evolved from this reaction path still has several sites for further oligomerization to higher hetero-oligomers.
On the basis of the similar product pattern of polymerization of FA by either Lignin peroxidase or HRP, it was suggested that FA oligomerization is governed by radical chemistry rather than enzyme specificity (15). These findings are in agreement with our data, which show that it is the chemistry of the enzymatically primarily formed radical products and especially the chemistry of the decarboxylated dehydrodimers of FA that leads to oligomerization. The GYG-FA hetero-oligomers found in the present study are clearly distinct from the ones found in a previous study (19). This shows that the type of oligomerization is strongly dependent on the substrate concentrations and kinetic control of the reaction.
Our present findings confirm the suggestion of Ward et al. (15) that the peroxidase-induced cross-linking of FA leads to higher oligomers than the trimers they identified by NMR. Furthermore, the attachment of higher oligomers of FA bound to a peptidyl tyrosine would supply a spatial basis for the covalent anchoring of easy diffusible small phenols on a tyrosine template in a plant cell wall protein and supports the often hypothesized template-guided in situ polymerization process of lignin (23)(24)(25). During growth or stress, increased levels of FA in the plant can result in differentiation of the type of tissue formed in a similar fashion as the kinetically and stoichiomet- rically controlled polymerization of FA described in this paper. The phenolic moiety enables oxidation of the molecule as a trigger for oxidative polymerization, whereas the vinylic moiety stabilizes the radical and allows further polymerization after decarboxylative dimerization.
Just as the peptide oligomers in a previous study always appeared to be blocked for further oligomerization by two nondecarboxylated FA molecules linked by dehydrogenation to the growing peptide oligomer (19), low concentrations of the FA monomer in plants can block possible sites for growth either on an esterified FA or on a protein-tyrosine template. High levels of FA statistically enhance dimerization to the decarboxylated FA dimers that enable further polymerization as established in the present study. The regulation of plant cell wall growth via control of the level of FA would therefore be amplified by an intrinsic feature of FA chemistry inevitably associated with FA concentration in the presence of peroxidases. We suggest that FA and possibly related cinnamic acid derivatives can exert two opposite effects on the growth of plant cell wall tissue, solely dependent on the incident concentration.