Synthetic Biology of Cannabinoids and Cannabinoid Glucosides in Nicotiana benthamiana and Saccharomyces cerevisiae.

Phytocannabinoids are a group of plant-derived metabolites that display a wide range of psychoactive as well as health-promoting effects. The production of pharmaceutically relevant cannabinoids relies on extraction and purification from cannabis (Cannabis sativa) plants yielding the major constituents, Δ9-tetrahydrocannabinol and cannabidiol. Heterologous biosynthesis of cannabinoids in Nicotiana benthamiana or Saccharomyces cerevisiae may provide cost-efficient and rapid future production platforms to acquire pure and high quantities of both the major and the rare cannabinoids as well as novel derivatives. Here, we used a meta-transcriptomic analysis of cannabis to identify genes for aromatic prenyltransferases of the UbiA superfamily and chalcone isomerase-like (CHIL) proteins. Among the aromatic prenyltransferases, CsaPT4 showed CBGAS activity in both N. benthamiana and S. cerevisiae. Coexpression of selected CsaPT pairs and of CHIL proteins encoding genes with CsaPT4 did not affect CBGAS catalytic efficiency. In a screen of different plant UDP-glycosyltransferases, Stevia rebaudiana SrUGT71E1 and Oryza sativa OsUGT5 were found to glucosylate olivetolic acid, cannabigerolic acid, and Δ9-tetrahydrocannabinolic acid. Metabolic engineering of N. benthamiana for production of cannabinoids revealed intrinsic glucosylation of olivetolic acid and cannabigerolic acid. S. cerevisiae was engineered to produce olivetolic acid glucoside and cannabigerolic acid glucoside.

Modulation of activity and substrate binding modes by mutation of single and double subsites +1/+2 and -5/-6 of barley alpha-amylase 1.

Enzymatic properties of barley alpha-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites -5/-6 (Cys95-->Ala/Thr) and +1/+2 (Met298-->Ala/Asn/Ser) as well as in the double mutants, Cys95-->Ala/Met298-->Ala/Asn/Ser. Cys95-->Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, kcat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside (Cl-PNPG7), respectively, but fivefold to 20-fold higher Km. The Cys95-->Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95-->Ala and wild-type. Met298-->Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas kcat and kcat/Km for Cl-PNPG7 are < or = 30% and < or = 10% of wild-type, respectively. The activity of Cys95-->Ala/Met298-->Ala/Asn/Ser is 100-180% towards starch, and the kcat/Km is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG7, respectively, emphasizing the strong impact of the Cys95-->Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG7 and amylose/Cl-PNPG7 are 2.8- to 270-fold and 1.2- to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites -5 and +2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites +1 and +2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6"'-maltotriosyl-maltohexaose thus covering subsites -1 to +5, while productive binding of unbranched substrate involves subsites -3 to +3.

Structural, physicochemical, and pasting properties of starches from potato plants with repressed r1-gene.

The aim of this work was to investigate the effect on starch molecular and physicochemical properties of down regulation of the R1 protein in potato (Solanum tuberosum L. cv. "Dianella") tubers. Most prominent is a 90% suppression of the phosphate content in the isolated potato tuber starch. The amylopectin chain length distribution profile as determined by HPAEC/PAD was not affected, but the amylose content was increased in the most down-regulated plants. The pasting properties of the transgenic starch revealed a pronounced decrease in peak viscosity and increased setback viscosity as measured using a rapid Visco analyzer. The starch gels displayed an increased hardness and stickiness with a maximum at 1.7 nmol of Glc-6P mg-1 of starch compared to the control lines. At very low phosphate levels (1.4 nmol of Glc-6P mg-1 of starch), the gel hardness was decreased as a result of increased gel brittleness. The increase in gel brittleness is believed to be an effect of an increased proportion of free amylopectin blocklets in the starch as determined by SEC/RI. The possible links between the structural and physicochemical parameters are discussed.

The action of starch synthase II on 6"'-alpha-maltotriosyl-maltohexaose comprising the branch point of amylopectin.

The principle of using a chemically synthesized, well-defined branched oligosaccharide to provide a more detailed knowledge of the substrate specificity of starch synthase II (SSII) is demonstrated. The branched nonasaccharide, 6"'-alpha-maltotriosyl-maltohexaose, was investigated as a primer for particulate SSII using starch granules prepared from the low-amylose pea mutant lam as the enzyme source. The starch granule preparation from the lam pea mutant contains no starch synthases other than SSII and is devoid of alpha-amylase, beta-amylase and phosphorylase activity. SSII was demonstrated to catalyse a specific nonprocessive elongation of the nonreducing end of the shortest unit chain of 6"'-alpha-maltotriosyl-maltohexaose, i.e. the maltotriose chain. Maltotriose and maltohexaose, representing the two linear building units of the branched nonasaccharide, were also tested as primers for SSII. Maltotriose was elongated more efficiently than 6"'-alpha-maltotriosyl-maltohexaose and maltohexaose was used less efficiently. Compared to the surface exposed alpha-glucan chains of the granule bound amylopectin molecules, all three soluble oligosaccharides tested were poor primers for SSII. This indicates that in vivo, the soluble oligosaccharides supposedly released as result of amylopectin trimming reactions are not re-introduced into starch biosynthetic reactions via the action of the granule bound fraction of SSII.

Resistance to an herbivore through engineered cyanogenic glucoside synthesis.

The entire pathway for synthesis of the tyrosine-derived cyanogenic glucoside dhurrin has been transferred from Sorghum bicolor to Arabidopsis thaliana. Here, we document that genetically engineered plants are able to synthesize and store large amounts of new natural products. The presence of dhurrin in the transgenic A. thaliana plants confers resistance to the flea beetle Phyllotreta nemorum, which is a natural pest of other members of the crucifer group, demonstrating the potential utility of cyanogenic glucosides in plant defense.

Active oxygen produced during selective excitation of photosystem I is damaging not only to photosystem I, but also to photosystem II.

With the aim to specifically study the molecular mechanisms behind photoinhibition of photosystem I, stacked spinach (Spinacia oleracea) thylakoids were irradiated at 4 degrees C with far-red light (>715 nm) exciting photosystem I, but not photosystem II. Selective excitation of photosystem I by far-red light for 130 min resulted in a 40% inactivation of photosystem I. It is surprising that this treatment also caused up to 90% damage to photosystem II. This suggests that active oxygen produced at the reducing side of photosystem I is highly damaging to photosystem II. Only a small pool of the D1-protein was degraded. However, most of the D1-protein was modified to a slightly higher molecular mass, indicative of a damage-induced conformational change. The far-red illumination was also performed using destacked and randomized thylakoids in which the distance between the photosystems is shorter. Upon 130 min of illumination, photosystem I showed an approximate 40% inactivation as in stacked thylakoids. In contrast, photosystem II only showed 40% inactivation in destacked and randomized thylakoids, less than one-half of the inactivation observed using stacked thylakoids. In accordance with this, photosystem II, but not photosystem I is more protected from photoinhibition in destacked thylakoids. Addition of active oxygen scavengers during the far-red photosystem I illumination demonstrated superoxide to be a major cause of damage to photosystem I, whereas photosystem II was damaged mainly by superoxide and hydrogen peroxide.

Synthesis of 4'-O-acetyl-maltose and alpha-D-galactopyranosyl-(1-->4)-D-glucopyranose for biochemical studies of amylose biosynthesis.

The chemical synthesis of the title compounds as maltose analogs, in which the non-reducing end is modified by acetylation of the 4'-OH group or by reversing its configuration, is reported. For synthesis of the 4'-O-acetylated analog, beta-maltose was converted into its per-O-benzylated-4',6'-O-benzylidene derivative followed by removal of the benzylidene acetal function and selective silylation at C-6'. Acetylation at C-4' of the obtained silylated compound followed by removal of the benzyl ether protecting groups and subsequent desilylation afforded the desired analog. The other maltose analog was synthesized via the glycosidation reaction between the glycosyl donor, O-(2,3,4,6-tetra-O-benzyl-alpha/beta-D-galactopyranosyl)trichloroacetimidate and the glycosyl acceptor, phenyl 2,3,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside followed by removal of the phenylthio group and debenzylation to provide the desired analog.

Transgenic tobacco and Arabidopsis plants expressing the two multifunctional sorghum cytochrome P450 enzymes, CYP79A1 and CYP71E1, are cyanogenic and accumulate metabolites derived from intermediates in Dhurrin biosynthesis.

Novel cyanogenic plants have been generated by the simultaneous expression of the two multifunctional sorghum (Sorghum bicolor [L.] Moench) cytochrome P450 enzymes CYP79A1 and CYP71E1 in tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis under the regulation of the constitutive 35S promoter. CYP79A1 and CYP71E1 catalyze the conversion of the parent amino acid tyrosine to p-hydroxymandelonitrile, the aglycone of the cyanogenic glucoside dhurrin. CYP79A1 catalyzes the conversion of tyrosine to p-hydroxyphenylacetaldoxime and CYP71E1, the subsequent conversion to p-hydroxymandelonitrile. p-Hydroxymandelonitrile is labile and dissociates into p-hydroxybenzaldehyde and hydrogen cyanide, the same products released from dhurrin upon cell disruption as a result of pest or herbivore attack. In transgenic plants expressing CYP79A1 as well as CYP71E1, the activity of CYP79A1 is higher than that of CYP71E1, resulting in the accumulation of several p-hydroxyphenylacetaldoxime-derived products in the addition to those derived from p-hydroxymandelonitrile. Transgenic tobacco and Arabidopsis plants expressing only CYP79A1 accumulate the same p-hydroxyphenylacetaldoxime-derived products as transgenic plants expressing both sorghum cytochrome P450 enzymes. In addition, the transgenic CYP79A1 Arabidopsis plants accumulate large amounts of p-hydroxybenzylglucosinolate. In transgenic Arabidopsis expressing CYP71E1, this enzyme and the enzymes of the pre-existing glucosinolate pathway compete for the p-hydroxyphenylacetaldoxime as substrate, resulting in the formation of small amounts of p-hydroxybenzylglucosinolate. Cyanogenic glucosides are phytoanticipins, and the present study demonstrates the feasibility of expressing cyanogenic compounds in new plant species by gene transfer technology to improve pest and disease resistance.

The distribution of covalently bound phosphate in the starch granule in relation to starch crystallinity.

Five selected starches with a 60-fold span in their content of monoesterified starch phosphate were investigated with respect to distribution of glucose 6-phosphate and glucose 3-phosphate residues, amylopectin chain length distributions and gelatinisation properties. The distribution of starch phosphate in the starch granules was determined by preparation of Nägeli dextrins followed by quantitative 31P-nuclear magnetic resonance spectroscopy. Total starch phosphate content was positively correlated to the unit chain lengths of the amylopectin as well as to the chain lengths of the corresponding Nägeli dextrins. The major part (68-92%) of the total starch phosphate content was partitioned to the hydrolysed (amorphous) parts. Starch-bound glucose 6-phosphate per milligram of starch was 2-fold enriched in the amorphous parts, whereas phosphate groups bound at the 3-position were more evenly distributed. The gelatinisation temperatures of the native starches as determined by differential scanning calorimetry were positively correlated (R(2)=0.75) to starch phosphate content, while crystallinity (gelatinisation enthalpy) and crystal heterogeneity (endotherm peak width) showed no correlations to starch phosphate content. The relations between starch molecular structure, architecture and functional properties are discussed.

Cloning and expression of cytochrome P450 enzymes catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of cyanogenic glucosides in Triglochin maritima.

Two cDNA clones encoding cytochrome P450 enzymes belonging to the CYP79 family have been isolated from Triglochin maritima. The two proteins show 94% sequence identity and have been designated CYP79E1 and CYP79E2. Heterologous expression of the native and the truncated forms of the two clones in Escherichia coli demonstrated that both encode multifunctional N-hydroxylases catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the two cyanogenic glucosides taxiphyllin and triglochinin in T. maritima. This renders CYP79E functionally identical to CYP79A1 from Sorghum bicolor, and unambiguously demonstrates that cyanogenic glucoside biosynthesis in T. maritima and S. bicolor is catalyzed by analogous enzyme systems with p-hydroxyphenylacetaldoxime as a free intermediate. This is in contrast to earlier reports stipulating p-hydroxyphenylacetonitrile as the only free intermediate in T. maritima. L-3,4-Dihydroxyphenyl[3-(14)C]Ala (DOPA) was not metabolized by CYP79E1, indicating that hydroxylation of the phenol ring at the meta position, as required for triglochinin formation, takes place at a later stage. In S. bicolor, CYP71E1 catalyzes the subsequent conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile. When CYP79E1 from T. maritima was reconstituted with CYP71E1 and NADPH-cytochrome P450 oxidoreductase from S. bicolor, efficient conversion of tyrosine to p-hydroxymandelonitrile was observed.

Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin. Cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes.

The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of L-valine and L-isoleucine, respectively, to the corresponding oximes. Two full-length cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast, Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes L-valine as well as L-isoleucine consistent with the co-occurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k(c) value with L-valine as substrate than with L-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. Both CYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes.

The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase that catalyzes the last step in synthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor. Isolation, cloning, heterologous expression, and substrate specificity.

The final step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor is the transformation of the labile cyanohydrin into a stable storage form by O-glucosylation of (S)-p-hydroxymandelonitrile at the cyanohydrin function. The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase was isolated from etiolated seedlings of S. bicolor employing Reactive Yellow 3 chromatography with UDP-glucose elution as the critical step. Amino acid sequencing allowed the cloning of a full-length cDNA encoding the glucosyltransferase. Among the few characterized glucosyltransferases, the deduced translation product showed highest overall identity to Zea mays flavonoid-glucosyltransferase (Bz-Mc-2 allele). The substrate specificity of the enzyme was established using isolated recombinant protein. Compared with endogenous p-hydroxymandelonitrile, mandelonitrile, benzyl alcohol, and benzoic acid were utilized at maximum rates of 78, 13, and 4%, respectively. Surprisingly, the monoterpenoid geraniol was glucosylated at a maximum rate of 11% compared with p-hydroxymandelonitrile. The picture that is emerging regarding plant glucosyltransferase substrate specificity is one of limited but extended plasticity toward metabolites of related structure. This in turn ensures that a relatively high, but finite, number of glucosyltransferases can give rise to the large number of glucosides found in plants.

Chemical synthesis of 6"'-alpha-maltotriosyl-maltohexaose as substrate for enzymes in starch biosynthesis and degradation.

A branched nonasaccharide 6"'-alpha-maltotriosyl-maltohexaose was synthesised in 40 steps from D-glucose and maltose. Phenyl O-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O- (2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-2,3-di-O-benzyl-1-th io- beta-D-glucopyranoside and O-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O-(2,3,6-tri- O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-alpha, beta-D-glucopyranosyl trichloroacetimidate were coupled by a general condensation reaction to form the per-O-benzylated branched hexasaccharide phenyl thioglycoside. The phenylthio group of this compound was converted into a trichloroacetimidate, which was coupled with phenyl O-(2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O-(2,3,6-tri-O- benzyl-alpha-D-glucopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-1-thio-beta-D- glucopyranoside to afford the per-O-benzylated branched nonasaccharide phenyl thioglycoside. Replacement of the phenylthio group with a free OH-group followed by hydrogenolysis gave the desired product. The synthons reported for this synthesis constitute a versatile tool for the chemical synthesis of other complex carbohydrates.

Biosynthesis of cyanogenic glucosides in Triglochin maritima and the involvement of cytochrome P450 enzymes.

The biosynthesis of the two cyanogenic glucosides, taxiphyllin and triglochinin, in Triglochin maritima (seaside arrow grass) has been studied using undialyzed microsomal preparations from flowers and fruits. Tyrosine was converted to p-hydroxymandelonitrile with V(max) and K(m) values of 36 nmol mg(-1) g(-1) fresh weight and 0.14 mM, respectively. p-Hydroxyphenylacetaldoxime and p-hydroxyphenylacetonitrile accumulated as intermediates in the reaction mixtures. Using radiolabeled tyrosine as substrate, the radiolabel was easily trapped in p-hydroxyphenylacetaldoxime and p-hydroxyphenylacetonitrile when these were added as unlabeled compounds. p-Hydroxyphenylacetaldoxime was the only product obtained using microsomes prepared from green leaves or dialyzed microsomes prepared from flowers and fruits. These data contrast earlier reports (Hösel and Nahrstedt, Arch. Biochem. Biophys. 203, 753-757, 1980; and Cutler et al., J. Biol. Chem. 256, 4253-4258, 1981) where p-hydroxyphenylacetaldoxime was found not to accumulate. All steps in the conversion of tyrosine to p-hydroxymandelonitrile were found to be catalyzed by cytochrome P450 enzymes as documented by photoreversible carbon monoxide inhibition, inhibition by antibodies toward NADPH-cytochrome P450 oxidoreductase, and by cytochrome P450 inhibitors. We hypothesize that cyanogenic glucoside synthesis in T. maritima is catalyzed by multifunctional cytochrome P450 enzymes similar to CYP79A1 and CYP71E1 in Sorghum bicolor except that the homolog to CYP71E1 in T. maritima exhibits a less tight binding of p-hydroxyphenylacetonitrile, thus permitting the release of this intermediate and its conversion into triglochinin.

Granule-bound starch synthase I in isolated starch granules elongates malto-oligosaccharides processively.

Isoforms of starch synthase belonging to the granule-bound starch synthase I (GBSSI) class synthesize the amylose component of starch in plants. Other granule-bound isoforms of starch synthase, such as starch synthase II (SSII), are unable to synthesize amylose. The kinetic properties of GBSSI and SSII that are responsible for these functional differences have been investigated using starch granules from embryos of wild-type peas and rug5 and lam mutant peas, which contain, respectively, both GBSSI and SSII, GBSSI but not SSII and SSII but not GBSSI. We show that GBSSI in isolated granules elongates malto-oligosaccharides processively, adding more than one glucose molecule for each enzyme-glucan encounter. Granule-bound SSII can elongate malto-oligosaccharides, but has a lower affinity for these than GBSSI and does not elongate processively. As a result of these properties GBSSI synthesizes longer malto-oligosaccharides than SSII. The significance of these results with respect to the roles of GBSSI and SSII in vivo is discussed.

Substrate specificity of the cytochrome P450 enzymes CYP79A1 and CYP71E1 involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench.

The two multifunctional cytochrome P450 enzymes, CYP79A1 and CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench have been characterized with respect to substrate specificity and cofactor requirements using reconstituted, recombinant enzymes and sorghum microsomes. CYP79A1 has a very high substrate specificity, tyrosine being the only substrate found. CYP71E1 has less stringent substrate requirements and metabolizes aromatic oximes efficiently, whereas aliphatic oximes are slowly metabolized. Neither CYP79A1 nor CYP71E1 catalyze the metabolism of a range of different herbicides. The reported resistance of sorghum to bentazon is therefore not linked to the presence of CYP79A1 or CYP71E1. NADPH is a much better cofactor than NADH although NADH does support the entire catalytic cycle of both P450 enzymes. Km and Vmax values for NADPH when supporting CYP71E1 activity are 0.013 mM and 111 nmol/mg protein/s. For NADH, the corresponding values are 0. 3 mM and 42 nmol/mg protein/s. CYP79A1 is a fairly stable enzyme. In contrast, CYP71E1 is labile and prone to rapid denaturation at room temperature. CYP71E1 is isolated in the low spin form. CYP71E1 catalyzes an unusual dehydration reaction of an oxime to the corresponding nitrile which subsequently is C-hydroxylated. The oxime forms a peculiar reverse Type I spectrum, whereas the nitrile forms a Type I spectrum. Several compounds which do not serve as substrates formed Type I substrate binding spectra with the two P450 enzymes.

Metabolic engineering of p-hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 from Sorghum bicolor.

Glucosinolates are natural products in cruciferous plants, including Arabidopsis thaliana. CYP79A1 is the cytochrome P450 catalysing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin in sorghum. Both glucosinolates and cyanogenic glucosides have oximes as intermediates. Expression of CYP79A1 in A. thaliana results in the production of high levels of the tyrosine-derived glucosinolate p-hydroxybenzylglucosinolate, which is not a natural constituent of A. thaliana. This provides further evidence that the enzymes have low substrate specificity with respect to the side chain. The ability of the cyanogenic CYP79A1 to integrate itself into the glucosinolate pathway has important implications for an evolutionary relationship between cyanogenic glucosides and glucosinolates, and for the possibility of genetic engineering of novel glucosinolates.

Double triton X-114 phase partitioning for the purification of plant cytochromes P450 and removal of green pigments.

A double Triton X-114 phase partitioning procedure that separates plant cytochromes P450 from green pigments and provides an extract highly enriched in total cytochromes P450 has been developed. Upon phase partitioning in Triton X-114, plant cytochromes P450 have previously been found to partition to the pigmented detergent rich phase. These partitionings were carried out using phosphate buffer. We found that the partitioning of the cytochromes P450 could be shifted to a pigment-free Triton X-114 poor phase by changing the buffer component to borate. The protein extract containing the cytochromes P450 but devoid of green pigment was subjected to a second phase partitioning step before which the buffer was changed from borate to phosphate. This second phase partitioning step produced a Triton X-114-rich phase highly enriched in cytochromes P450 proteins compared to the microsomal starting material as monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cytochrome P450 reconstitution assays, and Western blotting. The yield of the double phase partitioning purification procedure is about 26% which is high compared to the yields obtained at similar stages of purification using column chromatography. The double phase partitioning procedure takes 3-4 h to complete. This is very fast compared to traditional purification schemes for cytochromes P450 which involve multiple of column chromatographic steps. Plant cytochromes P450 are labile, low abundant proteins that are difficult to isolate. The double Triton X-114 phase partitioning here reported thus constitutes a versatile, efficient purification procedure circumventing many of the problems previously encountered.

Photosystem I is an early target of photoinhibition in barley illuminated at chilling temperatures.

Light-induced damage to photosystem I (PSI) was studied during low-light illumination of barley (Hordeum vulgare L.) at chilling temperatures. A 4-h illumination period induced a significant inactivation of PSI electron transport activity. Flash-induced P700 absorption decay measurements revealed progressive damage to (a) the iron-sulfur clusters FA and FB, (b) the iron-sulfur clusters FA, FB, and FX, and (c) the phylloquinone A1 and the chlorophyll AO or P700 of the PSI electron acceptor chain. Light-induced PSI damage was also evidenced by partial degradation of the PSI-A and PSI-B proteins and was correlated with the appearance of smaller proteins. Aggravated photodamage was observed upon illumination of barley leaves infiltrated with KCN, which inhibits Cu,Zn-superoxide dismutase and ascorbate peroxidase. This indicates that the photodamage of PSI in barley observed during low-light illumination at chilling temperatures arises because the defense against active oxygen species by active oxygen-scavenging enzymes is insufficient at these specific conditions. The data obtained demonstrate that photoinhibition of PSI at chilling temperatures is an important phenomenon in a cold-tolerant plant species.

Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin.

A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450- (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG-was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained. Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH-cytochrome P450-reductase in L-alpha-dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis. In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction. CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450-reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.