Biochemistry and Occurrence of O-Demethylation in Plant Metabolism

Demethylases play a pivitol role in numerous biological processes from covalent histone modification and DNA repair to specialized metabolism in plants and microorganisms. Enzymes that catalyze O- and N-demethylation include 2-oxoglutarate (2OG)/Fe(II)-dependent dioxygenases, cytochromes P450, Rieske-domain proteins and flavin adenine dinucleotide (FAD)-dependent oxidases. Proposed mechanisms for demethylation by 2OG/Fe(II)-dependent enzymes involve hydroxylation at the O- or N-linked methyl group followed by formaldehyde elimination. Members of this enzyme family catalyze a wide variety of reactions in diverse plant metabolic pathways. Recently, we showed that 2OG/Fe(II)-dependent dioxygenases catalyze the unique O-demethylation steps of morphine biosynthesis in opium poppy, which provides a rational basis for the widespread occurrence of demethylases in benzylisoquinoline alkaloid metabolism

Expressed sequence tag analysis of khat (Catha edulis) provides a putative molecular biochemical basis for the biosynthesis of phenylpropylamino alkaloids

Khat (Catha edulis Forsk.) is a flowering perennial shrub cultivated for its neurostimulant properties resulting mainly from the occurrence of (S)-cathinone in young leaves. The biosynthesis of (S)-cathinone and the related phenylpropylamino alkaloids (1S,2S)-cathine and (1R,2S)-norephedrine is not well characterized in plants. We prepared a cDNA library from young khat leaves and sequenced 4,896 random clones, generating an expressed sequence tag (EST) library of 3,293 unigenes. Putative functions were assigned to > 98% of the ESTs, providing a key resource for gene discovery. Candidates potentially involved at various stages of phenylpropylamino alkaloid biosynthesis from L-phenylalanine to (1S,2S)-cathine were identified

Functional genomics reveals novel o-demethylases involved in the biosynthesis of codeine and morphine in opium poppy

Bibliography: p. 170-193Some pages are in colour.Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a versatile model system to study plant alkaloid metabolism. The biosynthesis of morphine and related alkaloids in opium poppy occurs via a complex, multistep pathway beginning with the amino acid tyrosine. Corresponding genes encoding many of the enzymes involved in morphine biosynthesis have been isolated. However, molecular clones are not yet available for some enzymes, and enzyme activity accounting for two key O-demethylation steps leading from thebaine to morphine has yet to be detected. As part of a functional genomics platform aimed at isolating new genes, 1H nuclear magnetic resonance (NMR) metabolite profiling was used to characterize six varieties of opium poppy exhibiting altered alkaloid accumulation profiles. Aqueous and chloroform extracts of six different opium poppy cultivars were subjected to chemometric analysis. Principal component analysis of the 1H NMR spectra for latex extracts clearly distinguished two varieties, including a low-alkaloid variety "P" and a high-thebaine, low-morphine cultivar "T." Loading plots confirmed that morphinan alkaloids contributed predominantly to the variance in latex extracts. Relatively few differences were found in the levels of other metabolites, indicating that the variation was specific for alkaloid metabolism. This finding provided a rational basis for a microarray-based, comparative transcriptomics approach, wherein the transcriptome of T poppy stem was compared with those of high-morphine cultivars. This study led to the isolation of thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM), which together represented the first identified 0-demethylases in the 2-oxoglutarate/Fe(II)-dependent dioxygenase family. It was shown that gene-specific silencing of T6ODM and CODM dramatically alters morphinan alkaloid profiles of opium poppy

Metabolic engineering of hydroxycinnamic acid amide in nictoiana tabacum

Bibliography: p. 86-101Some pages are in colour.Feruloyltyramine and 4-coumaroyltyramine participate in the defense of plants against pathogens through their extracellular peroxidative polymerization, which is thought to reduce cell wall digestibility or otherwise inhibit fungal growth. Hydroxycinnamoyl­CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) and tyrosine decarboxylase (TYDC) are purported to play key roles in the stress-induced regulation of tyramine­derived hydroxycinnamic acid amide (HCAAT) biosynthesis. Transgenic tobacco (Nicotiana tabacum cv Xanthi) was engineered to constitutively express tobacco THT. A T₁ plant over-expressing THT was crossbred with T₁ tobacco expressing opium poppy TYDC2 to produce a T₂ line with elevated THT and TYDC activities compared with wild type plants. The effects of an independent increase in TYDC or THT activity, or a dual increase in both TYDC and THT on the cellular pools of HCAAT pathway intermediates and the accumulation of soluble and cell wall-bound feruloyltyrarnine and 4-coumaroyltyramine were examined. In roots, THT and TYDC activities were not substantially altered in transgenic lines beyond their relatively high wild type levels; thus, no changes in HCAAT precursor or product levels were detected. In leaves, increased TYDC activity resulted in a larger cellular pool of tyramine, and lower levels of phenylalanine. In contrast, elevated THT activity reduced tyramine levels. HCAAT levels were low in healthy leaves, but were induced in response to wounding and accumulated around wound sites. Similarly, endogenous THT and TYDC activities were wound-induced. The initial rate of wound-induced HCAAT accumulation was highest in transgenic plants with elevated THT and TYDC activities. These results show that both THT and TYDC exert partial and synergistic control over the flux of intermediates involved in HCAAT biosynthesis under some conditions

Alkaloid binding to opium poppy major latex proteins triggers structural modification and functional aggregation

Opium poppy accumulates copious amounts of several benzylisoquinoline alkaloids including morphine, noscapine, and papaverine, in the specialized cytoplasm of laticifers, which compose an internal secretory system associated with phloem throughout the plant. The contiguous latex includes an abundance of related proteins belonging to the pathogenesis-related (PR)10 family known collectively as major latex proteins (MLPs) and representing at least 35% of the total cellular protein content. Two latex MLP/PR10 proteins, thebaine synthase and neopione isomerase, have recently been shown to catalyze late steps in morphine biosynthesis previously assigned as spontaneous reactions. Using a combination of sucrose density-gradient fractionation-coupled proteomics, differential scanning fluorimetry, isothermal titration calorimetry, and X-ray crystallography, we show that the major latex proteins are a family of alkaloid-binding proteins that display altered conformation in the presence of certain ligands. Addition of MLP/PR10 proteins to yeast strains engineered with morphine biosynthetic genes from the plant significantly enhanced the conversion of salutaridine to morphinan alkaloids.</p

Summary of the construction and assembly for three Illumina NGS libraries.

<p>Abbreviations: CED, <i>Catha edulis</i>; ESI, <i>Ephedra sinica</i>; SRA, short-read archive; CDS, coding sequence.</p><p>Summary of the construction and assembly for three Illumina NGS libraries.</p

Proposed biosynthetic routes leading from L-phenylalanine to amphetamine-type alkaloids in khat and <i>Ephedra sinica</i>.

<p>A CoA-independent, non-β-oxidative pathway of L-phenylalanine side chain-shortening is shown in blue, whereas a CoA-dependent, β-oxidative route is shown in purple. Benzaldehyde, benzoic acid and/or benzoyl-CoA undergo condensation with pyruvate, a reaction putatively catalyzed by a ThDP-dependent carboligase. 1-Phenylpropane-1,2-dione undergoes transamination to yield (<i>S</i>)-cathinone, which is reduced to cathine and (1<i>R</i>,2<i>S</i>)-norephedrine. <i>N</i>-Methylation is restricted to <i>Ephedra</i> spp. and does not occur in khat. Activity has been detected for enzymes highlighted in yellow, and corresponding genes are available for enzymes highlighted in green. Enzymes highlighted in red have not been isolated, although database mining revealed numerous potential candidates (Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119701#pone.0119701.t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119701#pone.0119701.t003" target="_blank">3</a>). Abbreviations: CoA, Coenzyme A; NAD(H), nicotinamide adenine dinucleotide; NADP(H), nicotinamide adenine dinucleotide phosphate. PAL, phenylalanine ammonia lyase; ThDP, thiamine diphosphate.</p