Fig 1.
Putative biosynthetic pathway of CAM [11–20].
The terpenoid precursor secologanin was biosynthesized from MVA and / or MEP pathway through more than five steps of enzymatic oxidation. The amine precursor tryptamine was originated from shikimate pathway to tryptophan and followed by decarboxylation reaction. More than five cytochrome P450 enzymes were predicted to be involved in the conversion from strictosidine to CAM. The possible cytochrome P450 enzymes were highlighted in bold and its reduction partner CPR was in red. The other enzymes involved in the biosynthesis of CAM were omitted for clarity.
Fig 2.
Multiple sequence alignment of the characterized plant CPRs and the deduced amino acid sequence of CamCPR using Clustal Omega multiple alignment tool (a) and schematic representation of the key domains of CamCPR (b).
The conserved membrane anchor, FMN-, FAD-, P450-, cytochrome c-, and NADPH-binding domains were highlighted in different colors. The identical amino acid residues within each domain were highlighted in the same color. The cytochrome P450 reductases shown here were CrCPR from Catharanthus roseus (X69791), AtCPR from Arabidopsis thaliana (NM_118585), NfCPR from Nothapodytes foetida (EU604540); GhCPR from Gossypium hirsutum (FJ719368), HpCPR from Hybrid poplar (AF302496), OsCPR from Oryza sativa (AF302496), and PcCPR from Petroselinum crispum (AF024635). ANC, membrane anchor sequence; FMN, flavin mononucleotide binding domain; P450, cytochrome P450 binding domain; FAD, flavin adenine dinucleotide binding domain; NADPH, nicotine amide dinucleotide phosphate binding domain.
Table 1.
List of primers used in the study.
Fig 3.
Phylogenetic tree of the cytochrome P450 reductase family members.
Homologous sequences were obtained using the National Center for Biotechnology Information search engine (http://www.ncbi.nim.bih.gov/). The GenBank accession numbers for the sequences were as follows: CrCPR (Catharanthus roseus, X69791); AtCPR1 (Arabidopsis thaliana, NM_118585); AtCPR2 (Arabidopsis thaliana, X66017); NfCPR1 (Nothapodytes foetida, EU604540); NfCPR2 (Nothapodytes foetida, EU604541); NfCPR3 (Nothapodytes foetida, EU604542); GhCPR1 (Gossypium hirsutum, FJ719368); GhCPR2 (Gossypium hirsutum, FJ719369); HpCPR1 (Hybrid poplar, AF302496); HpCPR2 (Hybrid poplar, AF302497); HpCPR3 (Hybrid poplar, AF302498); OsCPR (Oryza sativa Japonica, AF302496); PcCPR1 (Petroselinum crispum, AF024634); PcCPR2 (Petroselinum crispum, AF024635); EcCPR (Eschscholzia californica, U67186); HtCPR (Helianthus tuberosus, A75963); VrCPR (Vigna radiate, L07843); PsmCPR (Papaver somniferum, U67185); PstCPR (Pisum sativum, AF002698); VsCPR (Vicia sativa, Z26252); AtCPR (Aegilops tauschii, EMT14573); AmCPR (Ammi majus, AY532374); AaCPR (Artemisia annua, DQ984181); CeCPR (Centaurium erythraea, AY596976); GmCPR1 (Glycine max, XM_003549388); GmCPR2 (Glycine max, XM_003541568); GmCPR3 (Glycine max, XM_003522720); HaCPR (Hypericum androsaemum, AY520902); LjCPR (Lotus japonicas, AB433810); McCPR (Matricaria chamomilla, KJ004519); MtCPR1 (Medicago truncatula, XM_003602850); MtCPR2 (Medicago truncatula, XM_003610061); ObCPR (Ocimum basilicum, JX524270); PgCPR (Panax ginseng, KF486915); PfCPR (Perilla frutescens, GQ120439); PxCPR1 (Petunia x hybrid, DQ099544); PxCPR2 (Petunia x hybrid, DQ099545); PkCPR (Picrorhiza kurrooa, JN968968); PmCPR (Pseudotsuga menziesii, Z49767); RcCPR1 (Ricinus communis, XM_002514003); RcCPR2 (Ricinus communis, XM_002534418); SmCPR1 (Salvia miltiorrhiza, FR693803); SmCPR2 (Salvia miltiorrhiza, JX848592); SaCPR1 (Santalum album, KC842187); SaCPR2 (Santalum album, KC842188); SdCPR (Scoparia dulcis, KF306080); SsCPR (Solenostemon scutellarioides, AM980997); SrCPR (Stevia rebaudiana, DQ269454); TchCPR (Taxus chinensis, AY959320); TcsCPR (Taxus cuspidate, AY571340); TcCPR1 (Theobroma cacao, XM_007018173); TcCPR2 (Theobroma cacao, XM_007014206); TcCPR3 (Theobroma cacao, XM_007018174); TcCPR4 (Theobroma cacao, XM_007018175); TaCPR (Triticum aestivum, AJ303373); TuCPR (Triticum urartu, EMS66177); ZmCPR (Zea mays, EU955593); ZoCPR1 (Zingiber officinale, AB566408); ZoCPR2 (Zingiber officinale, AB566409); Phylogenetic analysis was performed using the MEGA package and neighbor-joining program (http://www.megasoftware.net). The scale bar indicates the phylogenetic distance calculated according to the number of differences.
Fig 4.
Overexpression, purification, and characterization of recombinant CamCPR.
a, SDS-PAGE analyses of CamCPR (I) and tCamCPR (II). M, protein ruler; Lanes 1–4, purified enzyme; Lane 5 in I, cell lysate; Lane 5 in II, whole cell; Lane 6 in I, whole cell; Lane 6 in II, cell lysate. The target band was indicated with an arrowhead. b, the UV spectrum of tCamCPR was measured in 50 mM tris-HCl buffer (pH 7.4). c, the effects of different buffers with various pH on the cytochrome c reducing activity of tCamCPR. d–f, the steady-state kinetic constants of recombinant CamCPR. g, HPLC-DAD analyses of the reaction mixture of CamCPR supported cinnamic acid 4-hydrxoylase activity. Panel I, the authentic p-coumaric acid (♦) and trans-cinnamic acid (•); The HPLC traces of the whole reaction containing the cell lysates (panel II), the cells (panel III), and the boiled cells (panel IV) of the recombinant tCamCPR and CYP73A25 as catalyst.
Table 2.
Specific activities of various CamCPRs toward cytochrome c (25 μM), in the presence of 25 μM of NADPH or NADH.
Values are presented as mean ± SE. ND, not detected.
Fig 5.
The predicted 3-D structure of CamCPR.
a, schematic FMN-, FAD-, NADPH-, and P450-binding domains of CamCPR, built by the toolkit from PHYRE2 server; b, the amino acid residues involved in the binding of ligands (highlighted in greenish) such as FAD and NADPH, predicted by the 3DLigandSite tool; c, functional and structural residues of CamCPR with high scores were highlighted in red; and d, superimposition of CamCPR with the template RnCPR.
Fig 6.
Quantitative real-time PCR of CamCPR.
a, the 20-day-old C. acuminata seedlings; b, the real-time PCR amplification curves of CamCPR using rtCamCPR-F and rtCamCPR-R primers (Table 1); c, the PCR products melting curves of CamCPR; and d, the relative transcript levels of CamCPR in different tissues. The relative transcript level of CamCPR in the roots was set as control. Values are reported as means with standard error bars of three independent biological samples.
Fig 7.
Amino acid residues alignment of CamCPR, caa_locus_6894, caa_locus_12198, and caa_locus_112450 using Clustal Omega multiple aligment tool.
The identical amino acid residues between CamCPR and caa_locus_6894 were highlighted in red, between CamCPR and caa_locus_12198 were in green, and between CamCPR and caa_locus_112450 were in blue.
Fig 8.
Enzymatic digestion and chemical degradation of tCamCPR.
The predicted cleavage sites of tCamCPR (a) and the detected peptide fragments by SDS-PAGE analysis (b). T, thrombin; H, Hydroxylamine; M, protein ruler; Lane 1, tCamCPR; Lane 2, thrombin; Enzyme digestion products of tCamCPR with 1 U / 50 μL (Lane 3) and 2 U / 50 μL (Lane 4) of thrombin were used [37]; Lanes 5 and 6, chemical degradation products of tCamCPR by hydroxylamine [38].