Heterologous expression of the plant proteins strictosidine synthase and berberine bridge enzyme in insect cell culture

Kutchan, Toni M.; Böck, A.; Dittrich, Heinz

doi:10.1016/s0031-9422(00)94763-0

Cited by 77 publications

(40 citation statements)

References 15 publications

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“…In fact, similar results have been reported in studies on other plant enzymes. For example, BBE, which is localized in vacuoles in plant cells (44), is reported to be secreted into the medium when expressed in insect cells (45). We now investigate the subcellular localization of THCA synthase in C. sativa cells.…”

Section: Discussionmentioning

confidence: 99%

The Gene Controlling Marijuana Psychoactivity

Sirikantaramas

Morimoto

Ishikawa

et al. 2004

Journal of Biological Chemistry

243

147

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⌬ 1 -Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of ⌬ 1 -tetrahydrocannabinol. We cloned a novel cDNA (GenBank TM accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis.

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Section: Discussionmentioning

confidence: 99%

The Gene Controlling Marijuana Psychoactivity

Sirikantaramas

Morimoto

Ishikawa

et al. 2004

Journal of Biological Chemistry

243

147

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“…The cloning of the gene encoding BBE from the California poppy (E. californica) paved the way for the heterologous expression of the enzyme in Saccharomyces cerevisiae and Spodoptera frugiperda (7,8). The latter expression system produced ϳ4 mg of active enzyme per liter of insect cell culture, allowing some basic characterization of its substrate specificity and spectroscopic properties (5).…”

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confidence: 99%

Biochemical Evidence That Berberine Bridge Enzyme Belongs to a Novel Family of Flavoproteins Containing a Bi-covalently Attached FAD Cofactor

Winkler

Hartner

Kutchan

et al. 2006

Journal of Biological Chemistry

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111

106

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Berberine bridge enzyme (BBE) is involved in the transformation of (S)-reticuline to (S)-scoulerine in benzophenanthridine alkaloid biosynthesis of plants. In this report, we describe the high level expression of BBE encoded by the gene from Eschscholzia californica (California poppy) in the methylotrophic yeast Pichia pastoris employing the secretory pathway of the host organism. Using a two-step chromatographic purification protocol, 120 mg of BBE could be obtained from 1 liter of fermentation culture. The purified protein exhibits a turnover number for substrate conversion of 8.2 s ؊1 . The recombinant enzyme is glycosylated and carries a covalently attached FAD cofactor. In addition to the previously known covalent attachment of the 8␣-position of the flavin ring system to a histidine (His-104), we could also demonstrate that a covalent linkage between the 6-position and a thiol group of a cysteine residue (Cys-166) is present in BBE. The major evidence for the occurrence of a bi-covalently attached FAD cofactor is provided by N-terminal amino acid sequencing and mass spectrometric analysis of the isolated flavin-containing peptide. Furthermore, it could be shown that anaerobic photoirradiation leads to cleavage of the linkage between the 6-cysteinyl group yielding 6-mercaptoflavin and a peptide with the cysteine residue replaced by alanine due to breakage of the C-S bond. Overall, BBE is shown to exhibit typical flavoprotein oxidase properties as exemplified by the occurrence of an anionic flavin semiquinone species and formation of a flavin N(5)-sulfite adduct.

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“…This was followed by an additional, homology-based screen and by random sequencing of the same cDNA library from Taxus cuspidata from which over two dozen closely related cytochrome P450 candidate clones were obtained (Jennewein et al 2004a;Jennewein et al 2004b). For functional assessment with taxoid precursors, these clones were expressed in Saccharomyces cerevisiae WAT11 cells that coexpress an Arabidopsis NADPH:cytochrome P450 reductase (Pompon et al 1996), and were screened by in vivo feeding of the transformed yeast cultures, or were expressed using the baculovirus-Spodoptera system (Kutchan et al 1994) from which microsomes were isolated and assayed.…”

Section: Approaching Taxane Cytochrome P450 Oxygenasesmentioning

confidence: 99%

Cytochrome P450 oxygenases of Taxol biosynthesis

2006

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Cytochrome P450 monooxygenases play a prominent role in the biosynthesis of the diterpenoid anticancer drug Taxol, as they appear to constitute about half of the 19 enzymatic steps of the pathway in yew (Taxus) species. A combination of classical biochemical and molecular methods, including cell-free enzyme studies and differential-display of mRNA-reverse transcription polymerase chain reaction (RT-PCR) combined with a homology-based searching and random sequencing of a cDNA library from induced T. cuspidata cells, led to the discovery of six novel cytochrome P450 taxoid (taxane diterpenoid) hydroxylases. These genes show unusually high sequence similarity with each other (>70%) but low similarity (<30%) to, and significant evolutionary distance from, other plant P450s. Despite their high similarity, functional analysis of these hydroxylases demonstrated distinctive substrate specificities responsible for an early bifurcation in the biosynthetic pathway after the initial hydroxylation of the taxane core at C5, leading into a biosynthetic network of competing, but interconnected, branches. The use of surrogate substrates, in cases where the predicted taxoid precursors were not available, led to the discovery of two core oxygenases, the 2α-and the 7β-hydroxylase. This general approach could accelerate the functional analysis of candidate cDNAs from the extant family of P450 genes to identify the remaining oxygenation steps of this complex pathway.

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Heterologous expression of the plant proteins strictosidine synthase and berberine bridge enzyme in insect cell culture

Cited by 77 publications

References 15 publications

The Gene Controlling Marijuana Psychoactivity

The Gene Controlling Marijuana Psychoactivity

Biochemical Evidence That Berberine Bridge Enzyme Belongs to a Novel Family of Flavoproteins Containing a Bi-covalently Attached FAD Cofactor

Cytochrome P450 oxygenases of Taxol biosynthesis

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