Tobacco Cytochrome Research Articles

Molecular mechanisms of herbicide-inducible gene expression of tobacco CYP71AH11 metabolizing the herbicide chlorotoluron

Tobacco cytochrome P450 (CYP) 71AH11 metabolized the herbicide chlorotoluron, and its mRNA level was increased in tobacco culture cells by the treatment of 2,4-D. In order to clarify molecular mechanisms of induced gene expression of CYP71AH11 by herbicide treatment, a 1574-bp 5′-flanking region of CYP71AH11 was cloned, ligated to the reporter β-glucuronidase (GUS) gene, and then transformed into tobacco plants. The GUS activity in the transgenic tobacco plants was highly induced by bromoxynil treatment, followed by 2,4-D. Chlorotoluron was slightly increased the GUS activity. The bromoxynil-increased GUS activity was partially repressed by the antioxidants, suggesting that reactive oxygen species may be involved in activation of the 5′-flanking region of CYP71AH11 by bromoxynil treatment. Deletion and mutation assays showed that the region CD (−1281 to −770bp from the start codon of CYP71AH11) was important, but not sufficient, for response to bromoxynil. Electrophoretic mobility shift assays and southwestern blotting revealed that the sequences AAAAAG, and GAACAAAC and GAAAATTC in the CD region were important for interaction to the nuclear proteins of <30 and ≈75kDa, respectively. Particularly, interaction between AAAAAG and <30kDa proteins was increased by bromoxynil treatment. These results gave a cue for understanding the bromoxynil-induced gene expression of CYP71AH11, which may contribute to herbicide tolerance and selectivity in crop plants.

Molecular Dynamics Analysis Reveals Structural Insights into Mechanism of Nicotine N-Demethylation Catalyzed by Tobacco Cytochrome P450 Mono-Oxygenase

CYP82E4, a cytochrome P450 monooxygenase, has nicotine N-demethylase (NND) activity, which mediates the bioconversion of nicotine into nornicotine in senescing tobacco leaves. Nornicotine is a precursor of the carcinogen, tobacco-specific nitrosamine. CYP82E3 is an ortholog of CYP82E4 with 95% sequence identity, but it lacks NND activity. A recent site-directed mutagenesis study revealed that a single amino acid substitution, i.e., cysteine to tryptophan at the 330 position in the middle of protein, restores the NND activity of CYP82E3 entirely. However, the same amino acid change caused the loss of the NND activity of CYP82E4. To determine the mechanism of the functional turnover of the two molecules, four 3D structures, i.e., the two molecules and their corresponding cys–trp mutants were modeled. The resulting structures exhibited that the mutation site is far from the active site, which suggests that no direct interaction occurs between the two sites. Simulation studies in different biological scenarios revealed that the mutation introduces a conformation drift with the largest change at the F-G loop. The dynamics trajectories analysis using principal component analysis and covariance analysis suggests that the single amino acid change causes the opening and closing of the transfer channels of the substrates, products, and water by altering the motion of the F-G and B-C loops. The motion of helix I is also correlated with the motion of both the F-G loop and the B-C loop and; the single amino acid mutation resulted in the curvature of helix I. These results suggest that the single amino acid mutation outside the active site region may have indirectly mediated the flexibility of the F-G and B-C loops through helix I, causing a functional turnover of the P450 monooxygenase.

Mutagenesis of residues 27 and 78 modulates heme orientation in cytochrome b5

A comparison of the primary sequences of the heme binding domains of bovine and rat microsomal cytochrome reveal differences at only six residues. These residues must therefore provide the origin for the observed variation in the ratio of the heme orientational isomers, the equilibrium constant of which ranges from ∼9 in the bovine protein to ∼1.6 for rat cytochrome b 5. Residues 7, 20, 21, and 30 are distant from the exposed heme edge whilst Leu 27 and Phe 78 are located close to different parts of the porphyrin macrocycle. 1H NMR spectra of the heme and heme ligand resonances of a recombinant tobacco cytochrome b 5 extending from Gly 1 to Lys 89 suggest, in combination with NMR data acquired for other forms of cytochrome b 5 and an inspection of their sequence homology, that the identity of residue 78 influences the relative ratios of heme isomers. The Gly 1-Lys 89 domain of tobacco cytochrome b 5 has two equally abundant heme orientational isomers but retains the leucine side chain at position 27 whilst phenylalanine 78 is replaced by tyrosine. A more direct role for residue 78 in modulating the heme ratio is shown by site directed mutagenesis of bovine microsomal cytochrome b 5 where the mutation Phe 78>Tyr shifts the equilibrium constant for the heme orientational isomers from 9 to 3.5. Whilst the ratio is clearly shifted towards that exhibited by the rat protein the incomplete transition suggested the involvement of other residues. The mutation of Leu 27>Val was shown to result in a slightly smaller change in ratios of each isomer (from 9 to 4.0). Together these results point to the importance of these residues in modulating the ratio of heme isomers.

Cloning and expression in Escherichia coli and Saccharomyces cerevisiae of a novel tobacco cytochrome P-450-like cDNA

A cDNA library constructed from poly(A) + RNA of tobacco BY2 cells treated with 2,4-dichlorophenoxyacetic acid was screened by using a synthetic oligonucleotide corresponding to the heme binding region of avocado CYP71A1. A cloned 2-kb cDNA designated as cTBP contained an open reading frame of 1593 by encoding a protein of molecular size of 58 916. The deduced amino acid sequence included a cysteine residue corresponding to fifth ligand of heme-Fe at 497th. The coding sequence was expressed under the control of tac promoter and rrnB terminator in Escherichia coli to yield 7 to 10 nmol P450 equivalent per litre of the culture in the presence of δ-aminolevulinic acid. The modified coding sequences in which NH2-terminal residues 2–25 were replaced by the NH2-terminal 18 amino acid residues of microsomal bovine CYP17 were also expressed under the control of ADH promoter and terminator in Saccharomyces cerevisiae to yield 29 and 30 pmol of P450 equivalent/mg protein in the microsomal fraction, respectively. On co-expression of each of the modified coding sequences and yeast NADPH-cytochrome P-450 oxidoreductase gene, the yeast microsomas exhibited 7-ethoxycoumarin O-deethylase activity. Based on these results, tobacco cTBP was found to encode a novel P450-like species with a monooxygenese activity related to xenobiotic metabolism.

Isolation of a cDNA encoding a cytochrome b5 specifically expressed in developing tobacco seeds.

Polymerase chain reaction (PCR) was used to amplify transcripts encoding cytochrome b5 from cDNA synthesised from RNA isolated from developing seeds of tobacco (Nicotiana tabacum L.). The sequence of the amplified products indicated that the clones encoded a second form of tobacco cytochrome b5, different from that previously characterised (Smith et al. 1994, Plant Mol Biol 25:527-537). Rapid amplification of cDNA ends (RACE)-PCR was used to amplify the 5' and 3' ends of the transcript. Northern blotting and RNAse protection assays of RNA samples isolated from different tobacco tissues indicated that this second cytochrome b5 form was expressed only in developing seeds. Therefore, it seems likely that this message is the product of a tobacco cytochrome b5 gene specifically expressed in seeds.

Purification and immunochemical characteristics of NADPH-cytochrome P-450 oxidoreductase from tobacco cultured cells

NADPH-cytochrome P-450 oxidoreductase (EC 1.6.2.4) was purified from the microsomal fraction of tobacco ( Nicotiana tabacum) BY2 cells by chromatography on two anion-exchange columns and 2′,5′ ADP-Sepharose 4B column. The purified enzyme showed a single protein band with a molecular weight of 79 kDa on SDS-PAGE and exhibited a typical flavoprotein redox spectrum, indicating the presence of an equimolar quantity of FAD and FMN. This enzyme followed Michaelis-Menten Kinetics with K m values of 24 μM for NADPH and 16 μM for cytochrome c. An in vitro reconstituted system of the purified reductase with a partially purified tobacco cytochrome P-450 preparation showed the cinnamic acid 4-hydroxylase activity at the rate of 14 pmol min −1nmol −1 P-450 protein and with a purified rabbit P-450 2C14 catalyzed N-demethylation of aminopyrine at the rate of 6 pmol min −1 lnmo −1 P-450 protein. Polyclonal antibodies raised against the purified reductase reacted with tobacco reductase but not with yeast reductase on Western blot analysis. Anti-yeast reductase antibodies did not react with the tobacco reductase. This result indicate that the tobacco reductase was immunochemically different from the yeast reductase. The anti-tobacco reductase antibodies totally inhibited the tobacco reductase activity, but not the yeast reductase. Also, Western blot analyses using the anti-tobacco reductase antibodies revealed that leaves, roots and shoots of Nicotiana tabacum plants contained an equal amount of the reductase protein. From these results, it was suggested that there are different antibody binding sites, which certainly participate in enzyme activity, between tobacco and yeast reductase.

タバコ植物における光合成活性 I

In order to investigate the photosynthetic activity of tobacco plants with special reference to the relationship between this activity and the content of cytochrome f, the method of extraction and some properties of this compound was studied.Cytochrome f was extracted from tobacco chloroplasts, using ammoniac ethanol solution containing Triton X-100. Tobacco cytochrome f had properties similar to those of parsley cytochrome f.The concentration of cytochrome f in the extract was 0.18 to 0.2μmoles and not affected by the variety of tobacco plants. As the concentration of chlorophyll of tobacco plants varied remarkably among the varieties, the ratio of chlorophyll/cytochrome f differed due to the difference of the starting materials.