Plumbago indica L. contains naphthoquinones that are derived from six acetate units. To characterize the enzyme catalyzing the first step in the biosynthesis of these metabolites, a cDNA encoding a ...type III polyketide synthase (PKS) was isolated from roots of P. indica. The translated polypeptide shared 47–60% identical residues with PKSs from other plant species. Recombinant P. indica PKS expressed in Escherichia coli accepted acetyl‐CoA as starter and carried out five decarboxylative condensations with malonyl coenzyme A (‐CoA). The resulting hexaketide was not folded into a naphthalene derivative. Instead, an α‐pyrone, 6‐(2′,4′‐dihydroxy‐6′‐methylphenyl)‐4‐hydroxy‐2‐pyrone, was produced. In addition, formation of α‐pyrones with linear keto side chains derived from three to six acetate units was observed. As phenylpyrones could not be detected in P. indica roots, we propose that the novel PKS is involved in the biosynthesis of naphthoquinones, and additional cofactors are probably required for the biosynthesis of these secondary metabolites in vivo.
Cobalt complexation was investigated in a suspension cell culture of the cobalt hyperaccumulator
Crotalaria cobalticola.
C. cobalticola cells were more tolerant towards cobalt ions than the ...suspension cells of the non-accumulators
Rauvolfia serpentina and
Silene cucubalus. While the concentration of various compounds increased in cells of
C. cobalticola challenged with cobalt ions, phytochelatin biosynthesis was not induced. Instead, the exposure to cobalt ions resulted in the increase of citrate and cysteine in cells. Size exclusion chromatography demonstrated the co-elution of cobalt and cysteine in
C. cobalticola cell extracts. A significant increase in cysteine was observed also in cells of
R. serpentina and
S. cucubalus when they were exposed to cobalt ions. These results suggest that free cysteine is involved in cobalt ion complexation in plant cells.
Cysteine and citric acid increased when suspension cells of the cobalt hyperaccumulator
Crotalaria cobalticola were challenged with cobalt ions. Size exclusion chromatography suggests that cysteine participates in cobalt complexation in plant cells.
The berberine bridge enzyme ((S)-reticuline:oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9) catalyzes the oxidative cyclization of the N-methyl moiety of (S)-reticuline into the ...berberine bridge carbon, C-8, of (S)-scoulerine. This is a reaction that has neither an equivalent in organic chemistry nor a parallel in nature. The uniqueness of this catalytic reaction prompted an in depth study that began with the isolation of the cDNA encoding the berberine bridge enzyme followed by the overexpression of this cDNA in insect cell culture. The heterologously expressed enzyme has herein been shown to contain covalently attached FAD in a molar ratio of cofactor to protein of 1:1.03. Site-directed mutagenesis and laser desorption time-of-flight mass spectrometry suggest that the site of covalent attachment is at His-104. The holoenzyme exhibited absorbance maxima at 380 and 442 nm and a fluorescence emission maximum at 628 nm (310 nm excitation). Enzymic transformation of a series of (S)-reticuline derivatives modified with respect to the stereochemistry at C-1 or in the aromatic ring substitution suggests that ring closure proceeds in two steps: formation of the methylene iminium ion and subsequent ring closure via an ionic mechanism.
Summary
The biosynthesis of complex alkaloids in plants involves enzymes that, due to high substrate specificity, appear to have evolved solely for a role in secondary metabolism. At least one class ...of these enzymes, the oxidoreductases, catalyze transformations that are in some cases difficult to chemically mimick with an equivalent stereo‐ or regiospecificity and yield. Oxidoreductases are frequently catalyzing reactions that result in the formation of parent ring systems, thereby determining the class of alkaloid that a plant will produce. The oxidoreductases of alkaloid formation are a potential target for the biotechnological exploitation of medicinal plants in that they could be used for biomimetic syntheses of alkaloids. Analyzing the molecular genetics of alkaloid biosynthetic oxidations is requisite to eventual commercial application of these enzymes. To this end, a wealth of knowledge has been gained on the biochemistry of select monoterpenoid indole and isoquinoline biosynthetic pathways, and in recent years this has been complemented by molecular genetic analyses. As the nucleotide sequences of the oxidases of alkaloid synthesis become known, consensus sequences specific to select classes of enzymes can be identified. These consensus sequences will potentially facilitate the direct cloning of alkaloid biosynthetic genes without the need to purify the native enzyme for partial amino acid sequence determination or for antibody production prior to cDNA isolation. The current state of our knowledge of the biochemistry and molecular genetics of oxidases involved in alkaloid biosynthesis is reviewed herein.
Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities. Several groups of Amaryllidaceae alkaloids including the ...hemanthamine- and crinine-type alkaloids show promise as anticancer agents. Two reduction reactions are required for the production of these compounds: the reduction of norcraugsodine to norbelladine and the reduction of noroxomaritidine to normaritidine, with the enantiomer of noroxomaritidine dictating whether the derivatives will be the crinine-type or hemanthamine-type. It is also possible for the carbon-carbon double bond of noroxomaritidine to be reduced, forming the precursor for maritinamine or elwesine depending on the enantiomer reduced to an oxomaritinamine product. Here, a short chain alcohol dehydrogenase/reductase that co-expresses with the previously discovered norbelladine 4'-Omethyltransferase from Narcissus sp. and Galanthus spp. was cloned and expressed in Escherichia coli. Biochemical analyses and x-ray crystallography indicates that this protein functions as a noroxomaritidine reductase that forms oxomaritinamine from noroxomaritidine through a carbon-carbon double bond reduction. The enzyme also reduces norcraugsodine to norbelladine with a 400-fold lower specific activity. These studies identify a missing step in the biosynthesis of this pharmacologically important class of plant natural products.
•Terpene production did not affect plant morphology in the field.•Enhanced terpene lines performed better in the field than original lines.•Comparable lines produced more terpene in the greenhouse ...than field.•Terpene producing lines maintained stable oil composition under all conditions.•Cadinene lines had comparable seed yield to wild-type; limonene lines had less.
Bioengineered lines of the low-input oil seed crop Camelina sativa (Camelina), augmented to accumulate the monoterpene (4S)-limonene, the sesquiterpene (+)-δ-cadinene, or the sesquiterpene (+)-5-epi-aristolochene in seed, were evaluated for two growing seasons under field conditions to determine performance of the introduced traits in an agricultural setup including the effects on overall plant fitness, and total seed yield. Field-grown Camelina plants were further compared to greenhouse-grown plants to evaluate commonalities and differences resulting from cultivation under either controlled or agriculturally relevant growth conditions. Morphological appearance and plant height differed marginally between transgenic and wild-type plants under both greenhouse and field conditions, indicating low impact of the terpene production traits toward overall plant fitness. Total seed yield, however, was independent of the growth conditions and was reduced in Camelina lines producing (4S)-limonene by 48% on average and by 30% on average for (+)-5-epi-aristolochene producing lines. Conversely, (+)-δ-cadinene producing Camelina seed yields remained wild-type equivalent. Additional investigations included seed terpene accumulation, seed oil amount, and seed fatty acid composition. Terpene accumulation was reduced up to 22% for field-grown plants as compared to greenhouse-grown plants. Seed oil amounts were similar under greenhouse and field conditions but were consistently lowered by 2–5% for terpene producing lines. Similarly, seed oil composition remained stable under both field and greenhouse conditions, but generally favored a more energy dense phenotype in terpene producing Camelina lines. Lastly, outcrossing of transgenic traits to adjacent wild-type Camelina plants was observed under field but not greenhouse conditions.
Summary
In cell suspension cultures of the meadow rue
Thalictrum tuberosum
, biosynthesis of the anti‐microbial alkaloid berberine can be induced by addition of methyl jasmonate to the culture ...medium. The activities of the four methyltransferases involved in the formation of berberine from L‐tyrosine are increased in response to elicitor addition. Partial clones generated by RT–PCR with methyltransferase‐specific primers were used as hybridization probes to isolate four cDNAs encoding
O
‐methyltransferases from a cDNA library prepared from poly(A)+ RNA isolated from methyl jasmonate‐induced cell suspension cultures of
T. tuberosum
. RNA gel blot hybridization indicated that the transcripts for the methyltransferases accumulated in response to addition of methyl jasmonate to the cell culture medium. The cDNAs were functionally expressed in
Spodoptera frugiperda
Sf9 cells and were shown to have varying and broad substrate specificities. A difference of a single amino acid residue between two of the enzymes was sufficient to alter the substrate specificity. The four cDNAs were expressed either as four homodimers or as six heterodimers by co‐infection with all possible combinations of the four recombinant baculoviruses. These 10 isoforms thus produced displayed distinct substrate specificities and in some cases co‐infection with two different recombinant baculoviruses led to the
O
‐methylation of new substrates. The substrates that were
O
‐methylated varied in structural complexity from simple catechols to phenylpropanoids, tetrahydrobenzylisoquinoline, protoberberine and tetrahydrophenethylisoquinoline alkaloids, suggesting that some biosynthetic enzymes may be common to both phenylpropanoid and alkaloid anabolism.
Deacetylipecoside synthase (DIS), the enzyme catalyzing the condensation of dopamine and secologanin to form the (
R)-epimer of deacetylipecoside, has been purified 570-fold from the leaves of
...Alangium lamarckii and partially characterized. The isolated enzyme is a single polypeptide with
Mr 30, 000, and has a pH optimum at 7.5 and a temperature optimum at 45°C. The apparent
K
m values for dopamine and secologanin are 0.7 and 0.9 mM, respectively. DIS exhibits high substrate specificity toward dopamine, whereas neither tyramine nor tryptamine are utilized. The enzyme activity is not inhibited by its substrate dopamine, but is inhibited by alangimakine and dehydroalangimakine with similar I
50 values of 10 μM. DIS presumably provides (
R)-deacetylipecoside for the formation of tetrahydroisoquinoline monoterpene glucosides that also possess an (
R)-configuration at the same chiral center.
Three cDNAs encoding very similar but unique isoforms of chalcone synthase (EC 2.3.1.74) were isolated from a cDNA library prepared from RNA from root tissue of the Thai medicinal plant Cassia alata ...L. (ringworm bush, Leguminosae). Gene transcript for these three type-III polyketide synthases was found to accumulate predominantly in roots. The heterologously expressed enzymes accepted acetyl-, n-butyryl-, isovaleryl-, n-hexanoyl-, benzoyl-, cinnamoyl-, and p-coumaroyl-CoA as starter molecules and together with the co-substrate malonyl-CoA, formed multiple products. With the exception of the assay in which acetyl-CoA was used as the starter molecule, all substrates yielded a phloroglucinol derivative resulting from three sequential condensations of acetate units derived from three malonyl-CoA decarboxylations. Every substrate tested also produced two pyrone derivatives, one resulting from two acetate unit condensations (a bis-noryangonin-type pyrone derailment product) and one resulting from three acetate unit condensations (a 4-coumaroyltriacetic acid lactone-type pyrone derailment). C. alata accumulates the flavonoids quercetin, naringenin and kaempferol in roots, suggesting that the in planta function of these enzymes is the biosynthesis of root flavonoids.
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