Cloning and molecular characterization of the basic peroxidase isoenzyme from Zinnia elegans, an enzyme involved in lignin biosynthesis

Abstract

The major basic peroxidase from Zinnia elegans (ZePrx) suspension cell cultures was purified and cloned, and its properties and organ expression were characterized. The ZePrx was composed of two isoforms with a M(r) (determined by matrix-assisted laser-desorption ionization time of flight) of 34,700 (ZePrx34.70) and a M(r) of 33,440 (ZePrx33.44). Both isoforms showed absorption maxima at 403 (Soret band), 500, and 640 nm, suggesting that both are high-spin ferric secretory class III peroxidases. M(r) differences between them were due to the glycan moieties, and were confirmed from the total similarity of the N-terminal sequences (LSTTFYDTT) and by the 99.9% similarity of the tryptic fragment fingerprints obtained by reverse-phase nano-liquid chromatography. Four full-length cDNAs coding for these peroxidases were cloned. They only differ in the 5'-untranslated region. These differences probably indicate different ways in mRNA transport, stability, and regulation. According to the k(cat) and apparent K(m)(RH) values shown by both peroxidases for the three monolignols, sinapyl alcohol was the best substrate, the endwise polymerization of sinapyl alcohol by both ZePrxs yielding highly polymerized lignins with polymerization degrees > or =87. Western blots using anti-ZePrx34.70 IgGs showed that ZePrx33.44 was expressed in tracheary elements, roots, and hypocotyls, while ZePrx34.70 was only expressed in roots and young hypocotyls. None of the ZePrx isoforms was significantly expressed in either leaves or cotyledons. A neighbor-joining tree constructed for the four full-length cDNAs suggests that the four putative paralogous genes encoding the four cDNAs result from duplication of a previously duplicated ancestral gene, as may be deduced from the conserved nature and conserved position of the introns.

Figure 1.

Basic peroxidase isoenzyme patterns obtained by IEF under nonequilibrium conditions of the spent medium fraction of transdifferentiating 3-d-old Z. elegans mesophyll cell cultures (TEs), intercellular washing fluids obtained from 26-d-old hypocotyls and stems, and the spent medium fraction from SCC. Peroxidase isoenzymes were stained with 4-methoxy-α-naphthol and H₂O₂.

Figure 2.

Purification of the basic Z. elegans peroxidase isoenzyme from the spent medium of SCCs in a four-step purification protocol, which includes adsorption chromatography on phenyl Sepharose (A), size-exclusion chromatography on Superdex75 (B), cationic exchange chromatography on SP Sepharose (C), and affinity chromatography on concanavalin A (D). This last step resolves the Z. elegans basic peroxidase isoenzyme in two isoforms: one isoform, fully glycosylated and weakly retained by the column (D, peak a), and another isoform, partially glycosylated, and strongly retained by the column (D, peak b). Profiles of peroxidase activity and protein are denoted by a continuous and broken line, respectively.

Figure 3.

Protein fingerprints in SDS-PAGE at indicated steps during purification of the two isoforms of ZePrxs. From left to right, Molecular mass markers, whose values (×10⁻³) are drawn in the margin; crude extract protein (25 μg); protein after chromatography on phenyl Sepharose (1 μg), Superdex 75 (1 μg), and SP Sepharose (0.5 μg); and fully glycosylated isoform (0.25 μg; peak a in Fig. 2D) and partially glycosylated isoform (0.25 μg; peak b in Fig. 2D) after affinity chromatography on concanavalin A.

Figure 4.

SDS-PAGE of the glycosylated isoforms ZePrx34.70 (0.5 μg) and ZePrx33.44 (0.5 μg), and the deglycosylated isoforms d-ZePrx34.70 (6.4 μg) and d-ZePrx33.44 (5.3 μg), of the basic Z. elegans peroxidase isoenzyme after deglycosylation with TFMS.

Figure 5.

MALDI-TOF MS of tryptic (glyco-) peptides released from (A) the fully glycosylated isoform (ZePrx34.70) and (B) the partially glycosylated isoform (ZePrx33.44) of the basic Z. elegans peroxidase isoenzyme. Insets show in an amplified scale the tryptic fragments for both isoforms that were sequenced (arrowheads).

Figure 6.

cDNAs encoding both ZePrx33.44 and ZePrx34.70 obtained from RNA isolated from 6-d-old Z. elegans hypocotyls and deduced amino acid sequence of ZePrxs. The complete full-length sequence of the cDNAs, including the 5′ and 3′ flanking regions, was obtained by 5′-RACE and 3′-RACE using the primers RCPxZe-R (5′-RACE) and RCPxZe-L (3′-RACE; shaded in green). These cDNAs isolated from Z. elegans may be categorized as full-length cDNAs from the unusual length of the 5′ tails (216–245 bp), and by the fact that DNA transcription usually starts at a purine base (i.e. A), coding T (shaded in red) in the corresponding cDNAs. They also contain an identical poly(A) tract added to the 3′ end, just downstream of the signal sequence 5′-AATAAA-3′ (shaded in gray), which is that recognized by the poly(A) polymerase. The four full-length cDNAs contain an identical 966-bp ORF, flanked by an initiation (ATG) and a stop (TAA) codon, which translate into an identical amino acid sequence despite the observed C/T, G/A, G/C, and A/C substitutions (shaded in blue). Initiation of translation was established in the first (5′ proximal) ATG codon, since it was reinforced by the presence of an A at −3 (shaded in black). The four genes codifying these cDNAs consisted of three exons and two introns. The position in which each exon was disrupted by the introns was the same in the four genes: intron of type 2 is inserted −734 bp upstream of the end codon (black arrowhead), while intron of type 3 is inserted −389 bp upstream of the end codon (white arrowhead). The mature polypeptide starts with a Q residue, which probably generates the Z residue found in the purified enzyme. The predicted polypeptide also contains a peroxidase active site signature (33-AALVIRLLFHDC, shaded in green), a peroxidase proximal hemo-ligand signature (157-EMVALSGSHTL, shaded in green), eight conserved Cys (C11, C44, C49, C87, C93, C172, C198, C287, shaded in red), which probably yield the four disulfide bridges (C11-C87, C44-C49, C93-C287, C172-C198) common in most class III plant peroxidases, two putative N-glycosylation sites (181-NSTL and 191-NRSL, shaded in yellow), and two Ca²⁺-binding sites (shaded in blue), one distal (D43 and D50) and the other proximal (D211 and D219), characteristics for all the active class III peroxidases. The amino acid sequence common to the root RP5a peroxidase (Sato et al., 1995) and the nucleotide base sequence common to the expressed sequence tag (AJ504423) expressed by transdifferentiating Z. elegans mesophyll cell cultures, and described by Milloni et al. (2002), are underlined. Amino acid sequences matched by tryptic fragments and the experimentally determined N-terminal sequence are double underlined.

Figure 7.

A, pH-dependence profile. B, Temperature-dependence profile of the activity of ZePrx34.70 (○) and ZePrx33.44 (•) assayed against sinapyl alcohol. In A, peroxidase activity was measured in 50 mm Tris-acetate buffers of variable pH, containing 75 μm sinapyl alcohol and 50 μm H₂O₂. In B, peroxidase activity was measured in 50 mm Tris-acetate buffer, pH 5.0, containing 75 μm sinapyl alcohol and 50 μm H₂O₂. Values are means ± se (n = 3).

Figure 8.

GP-HPLC profiles of the products of sinapyl alcohol polymerization by ZePrx34.70 (A) and ZePrx33.44 (B), using the endwise protocol, which resolved them into a polymer of M_r ≥ 18,226 (peak 1, polymerization degree, n ≥ 87, λ_max = 278 nm), and into oligomers with mean M_r values of 4,052 (peak 2, n ≈ 19, λ_max = 283 nm), 1,274 (peak 3, n ≈ 6, λ_max = 283 nm), and 667 (peak 4, n ≈ 3, λ_max = 309 nm). Peak 5 (n ≈ 1, λ_max = 278 nm) was unreacted sinapyl alcohol.

Figure 9.

A, Molecular mass markers, whose values (×10⁻³) are drawn in the margin, and SDS-PAGE western blots using anti-ZePrx34.70 IgGs of ZePrx34.70 (1.0 μg) and ZePrx33.44 (1.0 μg). B, SDS-PAGE western blots using anti-ZePrx34.70 IgGs of total protein (10 μg) obtained from the spent medium fraction of Z. elegans SCC before (lane a) and after (lane b) treatment of blotted membranes with sodium periodate. C, SDS-PAGE western blots using anti-ZePrx34.70 IgGs of total protein obtained from the spent medium fraction of transdifferentiating 3-d-old Z. elegans TE (10 μg protein), cotyledons (500 μg protein), and leaves (250 μg protein), after treatment of the blotted membranes with sodium periodate. D, SDS-PAGE western blots using anti-ZePrx34.70 IgGs of 3-d-old (110 μg protein) and 6-d-old (120 μg protein) roots; 3-d-old (170 μg protein), 6-d-old (80 μg protein), and 26-d-old (2.5 μg protein) hypocotyls; and 6-d-old (200 μg protein) and 26-d-old (3 μg protein) stems, after treatment of the blotted membranes with sodium periodate.