Library of Norcoclaurine Synthases and Their Immobilization for Biocatalytic Transformations.

Norcoclaurine synthases (NCS), catalyzing a Pictet-Spengler reaction in plants as one of the first enzymes in the biosynthetic benzylisoquinoline pathway, are investigated for biocatalytic transformations. The library of NCS available is extended by two novel NCSs from Argemone mexicana (AmNCS1, AmNCS2) and one new NCS from Corydalis saxicola (CsNCS); furthermore, it is shown that the NCS from Papaver bracteatum (PbNCS) is a highly productive catalyst leading to the isoquinoline product with up to >99% e.e. Under certain conditions lyophilized whole Escherichia coli cells containing the various overexpressed NCS turned out to be suitable catalysts. The reaction using dopamine as substrate bears several challenges such as the spontaneous non-stereoselective background reaction and side reactions. The PbNCS enzyme is successfully immobilized on various carriers whereby EziG3 proved to be the best suited for biotransformations. Dopamine showed limited stability in solution resulting in the coating of the catalyst over time, which could be solved by the addition of ascorbic acid (e.g., 1 mg ml-1 ) as antioxidant.

Yeast extract induction of sanguinarine biosynthesis is partially dependent on the octadecanoic acid pathway in cell cultures of Argemone mexicana L., the Mexican poppy.

OBJECTIVE: To analyze the involvement of the octadecanoic (OCDA) pathway in the accumulation of sanguinarine induced by yeast extract (YE) in cell suspension cultures of Argemone mexicana (Papaveraceae). RESULTS: Exposure to YE promoted sanguinarine accumulation. This was not observed when they were exposed to methyl jasmonate (MeJa). Use of diethyldithiocarbamic acid (DIECA), an inhibitor of the OCDA pathway, resulted in partial impairment of this response. Exogenous application of MeJa did not reverse this effect in DIECA-exposed cultures. qRT-PCR revealed that the accumulation of transcripts corresponding to the berberine bridge enzyme gene, which was induced by YE exposure, was blocked by OCDA pathway and reversed by exogenous MeJa. Interestingly, this response pattern could not be observed on dihydrobenzophenanthridine oxidase enzyme activity, which was promoted by YE, but unaffected by either OCDA or MeJa. CONCLUSION: Results suggest partial involvement of OCDA pathway in this response.

Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells.

Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling.

Characterization of two methylenedioxy bridge-forming cytochrome P450-dependent enzymes of alkaloid formation in the Mexican prickly poppy Argemone mexicana.

Formation of the methylenedioxy bridge is an integral step in the biosynthesis of benzo[c]phenanthridine and protoberberine alkaloids in the Papaveraceae family of plants. This reaction in plants is catalyzed by cytochrome P450-dependent enzymes. Two cDNAs that encode cytochrome P450 enzymes belonging to the CYP719 family were identified upon interrogation of an EST dataset prepared from 2-month-old plantlets of the Mexican prickly poppy Argemone mexicana that accumulated the benzo[c]phenanthridine alkaloid sanguinarine and the protoberberine alkaloid berberine. CYP719A13 and CYP719A14 are 58% identical to each other and 77% and 60% identical, respectively, to stylopine synthase CYP719A2 of benzo[c]phenanthridine biosynthesis in Eschscholzia californica. Functional heterologous expression of CYP719A14 and CYP719A13 in Spodoptera frugiperda Sf9 cells produced recombinant enzymes that catalyzed the formation of the methylenedioxy bridge of (S)-cheilanthifoline from (S)-scoulerine and of (S)-stylopine from (S)-cheilanthifoline, respectively. Twenty-seven potential substrates were tested with each enzyme. Whereas CYP719A14 transformed only (S)-scoulerine to (S)-cheilanthifoline (K(m) 1.9±0.3; k(cat)/K(m) 1.7), CYP719A13 converted (S)-tetrahydrocolumbamine to (S)-canadine (K(m) 2.7±1.3; k(cat)/K(m) 12.8), (S)-cheilanthifoline to (S)-stylopine (K(m) 5.2±3.0; k(cat)/K(m) 2.6) and (S)-scoulerine to (S)-nandinine (K(m) 8.1±1.9; k(cat)/K(m) 0.7). These results indicate that although CYP719A14 participates in only sanguinarine biosynthesis, CYP719A13 can be involved in both sanguinarine and berberine formation in A. mexicana.