*2.4. PpaPrx19 Shows High Affinity for Cinnamyl Alcohols*

The homology that PpaPrx19 shows with peroxidases reportedly involved in lignification, together with the reported presence of lignin-like polyphenols in *P. patens*, led us to characterize this peroxidase based on its preferred substrates, using different well-known

peroxidase substrates, including natural precursors of lignin monomers (Table 4). Ascorbic acid is a typical substrate for class I (ascorbate) peroxidases but is poorly oxidized by class III secretory peroxidases [33]. The oxidation of NADH by peroxidases has been associated with cell wall loosening [34]. IAA is an in vitro peroxidase substrate and it has been reported to be catalyzed in vivo in relation to cell growth [35]. Hydroxycinnamic acids such as ferulic acid can be incorporated into suberin [36] and ferulate can also lead to cross-linking of the cell wall [37]. Coniferyl and sinapyl alcohols are polymerized by apoplastic peroxidases to form lignin [38]. Results showed that PpaPrx19 is able to use each assayed substrate, except ascorbic acid and NADH, although IAA was a poor substrate for PpaPrx19. This peroxidase is able to oxidize ferulic acid (0.53 ± 0.07 nkat <sup>μ</sup>g−<sup>1</sup> protein) and sinapyl alcohol (0.08 ± 0.01 nkat <sup>μ</sup>g−<sup>1</sup> protein) but the highest activity is shown using coniferyl alcohol as a substrate (1.46 ± 0.13 nkat <sup>μ</sup>g−<sup>1</sup> protein).

**Table 4.** Enzymatic activities (nkat μg−<sup>1</sup> protein) of purified peroxidase PpaPrx19 in the presence of different substrates. Data presented are average values ± SD of *n* = 3 experiments. n.d. not detected.


Trying to decipher a putative role in cinnamyl alcohol oxidation, we determined the catalytic parameters of PpaPrx19 for coniferyl and sinapyl alcohols. To calculate the kinetic constants, hydrogen peroxide was used at saturation levels (0.5 mM). The *K*<sup>M</sup> values were calculated according to Lineweaver–Burk equations. For PpaPrx19, apparent *K*<sup>M</sup> values were similar for both alcohols (16.7 μM for coniferyl alcohol and 20.8 μM for sinapyl alcohol). However, *K*cat is much higher for coniferyl alcohol, rendering a higher catalytic efficiency (*K*cat/*K*M), making coniferyl alcohol the best substrate (Table 5).

**Table 5.** Apparent *K*M, *Kcat* and *Kcat*/*K*<sup>M</sup> values for coniferyl alcohol (CA) and sinapyl alcohol (SA) shown by PpaPrx19.


With these extraordinary biochemical characteristics in mind, we searched for structural determinants that define a particular type of isoenzyme, the syringyl peroxidases [39]. We aligned PpaPrx19 with peroxidases with experimental capacity for oxidizing sinapyl alcohol, including ZePrx (the paradigmatic syringyl peroxidase); ATP A2 and HRP which are unable to oxidize sinapyl alcohol; and the three peroxidases that show the highest identity to PpaPrx19, as shown in Table 2. PpaPrx19 not only contains conserved residues important for catalytic mechanisms and the amino acids required for coordination of two Ca2+ ions (Figure 3), but it also presents most of the structural determinants of syringyl peroxidases (marked in red in Figure 3), which suggests that this peroxidase has no structural restrictions to oxidizing sinapyl alcohol [39]. As a matter of fact, the PpaPrx19 catalytic properties suggest this peroxidase shows a low *K*<sup>M</sup> for sinapyl alcohol and its ability to oxidize this substrate in vitro (Table 5).


**Figure 3.** PpaPrx19 shows most of residues characteristic of syringyl peroxidases. Amino acid alignment of mature peroxidase sequences, including those purified in this study (PpaPrx19), *Arabidopsis thaliana* ATP A2 (CAA68212), horseradish peroxidase HRPC1 (AAA33377), *Picea abies* PabPrx04 (CAH10839), *Zinnia elegans* ZePrx (CAI54302), *Solanum lycopersicum* TPX2 (AAA65636) and the three peroxidases that show the highest identity to PpaPrx19 (PpaPrx09, TruPrx01 and CppPrx02). Conserved residues important for catalytic mechanisms are shaded in green, the calcium-binding sites are shaded in gray, the S-S bridge-forming cysteines are shaded in yellow, putative *N*-glycosylation site of PpaPrx19 is shaded in blue and structural determinants of syringyl peroxidases are shaded in red. Consensus symbols: '\*' indicates fully conserved residues, ':' indicates conserved substitutions and '.' indicates semiconserved residue substitutions.
