Molecular cloning and characterization of a vacuolar class III peroxidase involved in the metabolism of anticancer alkaloids in Catharanthus roseus

Abstract

Catharanthus roseus produces low levels of two dimeric terpenoid indole alkaloids, vinblastine and vincristine, which are widely used in cancer chemotherapy. The dimerization reaction leading to alpha-3',4'-anhydrovinblastine is a key regulatory step for the production of the anticancer alkaloids in planta and has potential application in the industrial production of two semisynthetic derivatives also used as anticancer drugs. In this work, we report the cloning, characterization, and subcellular localization of an enzyme with anhydrovinblastine synthase activity identified as the major class III peroxidase present in C. roseus leaves and named CrPrx1. The deduced amino acid sequence corresponds to a polypeptide of 363 amino acids including an N-terminal signal peptide showing the secretory nature of CrPrx1. CrPrx1 has a two-intron structure and is present as a single gene copy. Phylogenetic analysis indicates that CrPrx1 belongs to an evolutionary branch of vacuolar class III peroxidases whose members seem to have been recruited for different functions during evolution. Expression of a green fluorescent protein-CrPrx1 fusion confirmed the vacuolar localization of this peroxidase, the exact subcellular localization of the alkaloid monomeric precursors and dimeric products. Expression data further supports the role of CrPrx1 in alpha-3',4'-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant.

Figure 1.

Biosynthesis of vinblastine from the monomeric precursors catharanthine and vindoline. Anhydrovinblastine is the product of the dimerization reaction and the precursor of the anticancer drugs.

Figure 2.

The gene and protein of peroxidase 1 from C. roseus (CrPrx1). A, Schematic representation of the gene (P, promoter; Int, intron; Ex, exon; UTR, untranslated region). B, Nucleotide and deduced amino acid sequence. The coding region is shown in uppercase letters. The signal peptide and the putative C-terminal propeptide are represented in italics. Sequences highlighted correspond to the peroxidase active site signature (positions 38–49) and to the peroxidase proximal heme-ligand signature (positions 168–178). The single amino acids highlighted are the eight conserved Cys, which allow the formation of four disulfide bridges. Sequences underlined in black match perfectly the sequences determined for the nine sequenced peptides (Table I). Sequences underlined in gray correspond to putative glycosylation sites. Boxes, aataaa polyadenylation signals. Arrowheads, Different positions of the poly(A) tail determined by RACE for seedlings. Arrows, Different positions of the poly(A) tail in two clones isolated from the cDNA library prepared from the first pair of leaves. Accession numbers are: AM236087 (mRNA), AM236088 (promoter region), and AM236089 (intron I).

Figure 3.

Southern blot of C. roseus genomic DNA. An antisense DIG-labeled oligonucleotide complementary to a 39-base segment of intron I was used as probe. DNA was digested with NcoI (lane 1), Van91I and EcoRI (lane 2), and HincII (lane 3). Arrows indicate bands, and numbers on the left indicate positions of the DNA M_r markers in kilobase pairs.

Figure 4.

Multiple alignment of CrPrx1 with other class III peroxidases. The sequences used include the most similar class III peroxidases (NtPer9-6, LePrx16, and VaPrx01), the most similar class III peroxidases found in Arabidopsis and rice genomes (ApPrx12 and OsPrx23), and the significantly similar and well-characterized barley BP1. The extensively characterized peanut PNC1 and horseradish HrPC (PrxC1A) are also included. Signal peptides and C-terminal propeptides determined experimentally are underlined. Arrowheads, Eight conserved Cys of class II peroxidases. 1, Highly conserved sequences present in all peroxidases of the same phylogenetic branch of CrPrx1 (Fig. 6). 2, Peroxidase active site signature (distal heme-ligand). 3, Peroxidase proximal heme-ligand signature. Accession numbers and percentages of identity with CrPrx1 are: tobacco NtPer9-6, AY032674, 75% identity; tomato LePrx16, TC124085 TIGR, 71% identity; adzuki bean VaPrx01, D11337 NCBI, 66% identity; Arabidopsis AtPrx12, locus At1g71695, 58% identity; barley BP1, TC132499 TIGR, 54% identity; rice OsPrx23, BN000552 NCBI, 53% identity; horseradish PrxC1A, M37156 NCBI, 41% identity; and peanut PNC1, M37636 NCBI, 42% identity (identities determined using the Swiss Institute of Bioinformatics Blast Network Service through Peroxibase, except for PNC1 and PrxC1, which were determined using NCBI Blast 2 sequences). Numbering of sequences starts at first amino acid translated, meaning that CrPrx1 amino acid 1 of Figure 2B corresponds to amino acid 35 in this image due to the 34 amino acids of the signal peptide.

Figure 5.

Hypothetical 3D structural model of CrPrx1. The model was constructed using the x-ray crystallography coordinates for barley peroxidase BP1. Colors represent surface charge: white, neutral; blue, positive; and red, negative. The pore leading to the centrally located heme can be observed on the left. The figure was prepared by homology modeling with barley peroxidase BP1 as the template, using SWISS-MODEL and the Swiss-Pdb Viewer (Guex and Peitsch, 1997), which are available at www.expasy.ch/spdbv/.

Figure 6.

Unrooted neighbor-joining phylogenetic tree relating CrPrx1 with other class III peroxidases. The following sequences retrieved from Peroxibase were used: (1) highly identical peroxidases including the most identical present in the genomes of Arabidopsis (AtPrx12) and rice; (2) extensively studied basic peroxidases (barley BP1, horseradish HrPC1, and peanut PNC1); and (3) six further Arabidopsis peroxidases, representing, together with AtPrx12, all the seven branches revealed in the phylogenetic tree of Arabidopsis peroxidases constructed by Tognolli et al. (2002). The tree was constructed using the ClustalW multiple sequence alignment tool of GenomeNet of the Kyoto University Bioinformatics Center, using default parameters (http://align.genome.jp/). See accession numbers of represented genes in legend of Figure 4. Nomenclature of Arabidopsis peroxidase genes according to class III Peroxibase (http://peroxidase.isb-sib.ch).

Figure 7.

Transient transformation of C. roseus cells with CrPrx1-GFP fusions. A, Scheme of constructs used for transformation. B, Typical GFP fluorescence pattern observed for transformation with each construct. 1a to 1c, 35S∷GFP, control for GFP localization. GFP accumulates in the cytoplasm and nucleus. 2a to 2c, 35S∷CrNTPP-GFP, construct with the NTPP of CrPrx1 (CrNTPP). GFP accumulates in the ER and possibly Golgi. 3a to 3e, 35S∷CrNTPP-GFP-CrPrx1, CrPrx1 does not include the NTPP sequence. GFP accumulates in the central vacuole. 4a, 35S∷CrNTPP-GFP-CrPrx1ΔCTE, the same as 3 but excluding the sequence of the C-terminal extension. GFP accumulates in the ER and possibly Golgi. 5a to 5c, 35S∷SP-GFP-ChitA, positive control for vacuolar localization of GFP, with the signal peptide from pumpkin 2S albumin (SP in image) and the C-terminal propeptide of tobacco chitinase A (ChitA in image), a confirmed vacuolar sorting signal (Tamura et al., 2003). GFP accumulates in the vacuole. Bar = 20 μm.

Figure 8.

mRNA accumulation of CrPrx1, Str, and D4h in different organs and leaf developmental stages of C. roseus. Str codes for an enzyme of the beginning of the indole alkaloid pathway and D4h codes for an enzyme of a later part of the indole alkaloid pathway, near the dimerization step. 1st, First pair of leaves; FD, fully developed leaves; PS, presenescent leaves; S, senescent leaves; Pet, petals; RO, reproductive organs; R, roots. For identification of developmental stages, see Figure 9A. Duplicate results are presented.

Figure 9.

A, Scheme illustrating leaf developmental stages. B, Regulation of CrPrx1 gene expression and CrPrx1 activity levels in different leaf developmental stages. C. roseus plants were 1 year old. [See online article for color version of this figure.]

Figure 10.

Accumulation of alkaloids and CrPrx1 at different leaf developmental stages. A, Levels of catharanthine, vindoline, and AVLB from the different leaf pairs of a single plant. The pattern is representative of four different plants analyzed. B, Activity of CrPrx1 assumed from total peroxidase activity of leaf protein extracts (mean of four plants). C, CrPrx1 band in IEF gels stained for peroxidase activity. Leaf pairs are numbered from the shoot tip. C. roseus plants were 16 weeks old.

Figure 11.

A, Time course of the dimerization reaction catalyzed by CrPrx1. The reaction mixture contained 2 μm CrPrx1, 300 μm vindoline, 400 μm catharanthine, and 330 μm H₂O₂ in 0.1 m MES, pH 6.8. B, HPLC chromatograms of the substrates of the dimerization reaction (0 min) and of the time point corresponding to a maximum yield of the dimerization reaction (30 min). V, Vindoline; C, catharanthine.