BERBERINE PowerPoint PPT Presentations ..
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Goldenseal Rich in alkaloids berberine, hydrastine Potential ..
A major unresolved aspect of noscapine biosynthesis concerns the transformation of the protoberberine to the secoberbine backbone as a component in the ultimate formation of the phthalideisoquinoline scaffold. On structural grounds, secoberbine alkaloids, such as macrantaline and macrantoridine, were considered as possible intermediates between a protoberberine precursor, such as scoulerine or 1-methoxyscoulerine, and noscapine (). An alternative proposal involves the stereospecific N-methylation of scoulerine followed by multistage oxidation to egenine, which is further oxidized to phthalideisoquinoline alkaloids, such as bicuculline (). A protoberberine, such as scoulerine, has also been hypothesized to potentially undergo N-methylation and C8 oxidation to the secoberbine macrantaldehyde, which could be reduced to macrantaline or oxidized to macrantoridine (). Theoretically, macrantaldehyde could undergo oxidation to narcotinehemiacetal (). As such, a hypothetical scheme for noscapine biosynthesis in opium poppy was proposed whereby N-methylcanadine is hydroxylated at C1 and possibly O-methylated to form 1-methoxy-N-methylcanadine (). Oxidation of 1-methoxy-N-methylcanadine at C8 was seen as the entry point to the formation of a secoberbine intermediate, such as macrantaldehyde. The drawback of this hypothesis is the lack of empirical evidence for the occurrence of secoberbine alkaloids in opium poppy. Most known secoberbine alkaloids have been isolated from different Papaver species such as Papaver pseudo-oriental, Papaver armeiacum, and Papaver fugax (Sariyar 2002). However, the proposed pathway extrapolated from the partial physiological characterization of a putative noscapine biosynthetic gene cluster supports the natural occurrence of secoberbine alkaloids in opium poppy (). The individual suppression of transcript levels for six of ten clustered genes using VIGS was interpreted to suggest a biosynthetic route involving an initial ring opening (i.e., cleavage of the berberine bridge) on N-methylcanadine catalyzed by either CYP82X1 or CYP82Y1 and yielding a secoberbine intermediate. CYP82X2 was proposed to then catalyze hydroxylation at the C3 position, yielding a derivative of this secoberbine intermediate. The two schemes agree on the involvement of an enzymatic product derived from N-methylcanadine as the entry point to unidentified secoberbine intermediates in noscapine biosynthesis.
- Benzylisoquinoline alkaloid biosynthesis.
Three CYP families have been implicated in BIA metabolism (). Whereas several enzymes in the CYP80 and CYP719 families have been shown to catalyze aromatic hydroxylation and inter- and intramolecular C-O and C-C coupling reactions involved in 1-benzylisoquinoline, protoberberine, aporphine, bisbenzylisoquinoline, and morphinan alkaloid biosynthesis, only two members in the CYP82 family have been functionally characterized: CYP82N4 catalyzes aliphatic hydroxylation at C14 of N-methylcanadine and N-methylstylopine, resulting in tautomerization to allocryptopine and protopine, respectively (), whereas CYP82N2v2 from E. californica catalyzes aliphatic hydroxylation at C6 of protopine and allocryptopine leading to intramolecular rearrangement to the benzo[c]phenenthridine scaffold (). CYP82Y1 displays considerable sequence identity with CYP82N4 (53%) and CYP82N2v2 (44%) and, similar to CYP82N4, also accepted only N-methylcanadine and N-methylstylopine as substrates. In contrast, CYP82Y1 and CYP82N4 catalyze regiospecific hydroxylation at C1 and C14, respectively. Also, CYP82N4 converted both N-methylstylopine and N-methylcanadine at similar rates (), whereas PsCYP82Y1 converted N-methylcanadine with greater efficiency than N-methylstylopine. Alterations in the residues responsible for substrate recognition and alkaloid binding might be attributed to differences in substrate preference and oxidation activity among CYP82 variants. In contrast with the KPIAPXXXPH substrate recognition site motif of enzymes in the CYP719 family, CYP82Y1, CYP82X1, and CYP82X2 contain a distinct YPA(G/S)XXX(E/D)R domain and a conserved Gly-370 residue. However, whereas the residues specific for alkaloid binding in N-methylstylopine 14-hydroxylase (CYP82N4) () are Ile and Leu, the corresponding residues in CYP82Y1 are Leu and Ser (). CYP82N2v2 was relatively promiscuous with protopine alkaloid substrates, including protopine, allocryptopine, 13-oxoprotopine, and corycavine (). Other substituted quaternary protoberberine alkaloids beyond N-methylcanadine and N-methylstylopine could also serve as substrates for CYP82Y1 and CYP82N4, although such reactions might not be physiologically relevant. A narrow substrate range is common among CYPs involved in the biosynthesis of BIAs (–) and other plant-specialized metabolites. For example, CYP82G1 from tobacco (N. tabacum), which catalyzes the final step in the biosynthesis of common homoterpene volatiles, displays substrate specificity for (E,E)-geranyllinalool and its C15 analog (E)-nerolidol (), and CYP82C2 and CYP82C4 from mouse ear cress (A. thaliana) show substrate specificity for 8-methoxypsoralen ().
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