Benzaldehyde is a precursor of phenylpropylamino alkaloids as revealed by targeted metabolic profiling and comparative biochemical analyses in Ephedra spp.

Phytochemistry 81 (2012) 71–79

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Benzaldehyde is a precursor of phenylpropylamino alkaloids as revealed by targeted metabolic profiling and comparative biochemical analyses in Ephedra spp. Raz Krizevski a,b, Einat Bar a, Or Shalit c, Asaf Levy a, Jillian M. Hagel d, Korey Kilpatrick e,f, Frédéric Marsolais e,f, Peter J. Facchini d, Shimon Ben-Shabat g, Yaron Sitrit h, Efraim Lewinsohn a,⇑ a

Department of Aromatic, Medicinal and Spice Crops, Newe Ya’ar Research Center, Agricultural Research Organization, P.O. Box 1021, Ramat Yishay 30095, Israel Albert Katz Department of Dryland Agriculture, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boqer 84990, Israel c Department of Biology, University of Haifa-Oranim, Tiv’on 36006, Israel d Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada e Department of Biology, Western University, London, Ontario, Canada f Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food, Canada g Department of Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel h The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva, Bergman Campus 84105, Israel b

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Article history: Received 2 November 2011 Received in revised form 13 May 2012 Accepted 14 May 2012 Available online 21 June 2012 Keywords: Ephedra sinica Stapf Ephedra foeminea Metabolic pathway elucidation Ephedrine alkaloids Benzaldehyde carboxyligase (S)-Cathinone reductase, (1R,2S)norephedrine N-methyltransferase

a b s t r a c t Ephedrine and pseudoephedrine are phenylpropylamino alkaloids widely used in modern medicine. Some Ephedra species such as E. sinica Stapf (Ephedraceae), a widely used Chinese medicinal plant (Chinese name: Ma Huang), accumulate ephedrine alkaloids as active constituents. Other Ephedra species, such as E. foeminea Forssk. (syn. E. campylopoda C.A. Mey) lack ephedrine alkaloids and their postulated metabolic precursors 1-phenylpropane-1,2-dione and (S)-cathinone. Solid-phase microextraction analysis of freshly picked young E. sinica and E. foeminea stems revealed the presence of increased benzaldehyde levels in E. foeminea, whereas 1-phenylpropane-1,2-dione was detected only in E. sinica. Soluble protein preparations from E. sinica and E. foeminea stems catalyzed the conversion of benzaldehyde and pyruvate to (R)-phenylacetylcarbinol, (S)-phenylacetylcarbinol, (R)-2-hydroxypropiophenone (S)-2hydroxypropiophenone and 1-phenylpropane-1,2-dione. The activity, termed benzaldehyde carboxyligase (BCL) required the presence of magnesium and thiamine pyrophosphate and was 40 times higher in E. sinica as compared to E. foeminea. The distribution patterns of BCL activity in E. sinica tissues correlates well with the distribution pattern of the ephedrine alkaloids. (S)-Cathinone reductase enzymatic activities generating (1R,2S)-norephedrine and (1S,1R)-norephedrine were significantly higher in E. sinica relative to the levels displayed by E. foeminea. Surprisingly, (1R,2S)-norephedrine N-methyltransferase activity which is a downstream enzyme in ephedrine biosynthesis was significantly higher in E. foeminea than in E. sinica. Our studies further support that benzaldehyde is the metabolic precursor to phenylpropylamino alkaloids in E. sinica. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Ephedrine alkaloids are important pharmacoactive constituents present in many Ephedra species acting as adrenergic agonists both by direct adrenaline agonistic activity as well as by indirect release of norepinephrine (Bruneton, 1995; Rothman et al., 2003). These unique group of natural products include (S)-cathinone, (1R,2S)norephedrine, (1S,2S)-norpseudoephedrine, (1R,2S)-ephedrine, (1S,2S)-pseudoephedrine, (1R,2S)-N-methylephedrine and (1S,2S)N-methylpseudoephedrine (Bruneton, 1995; Krizevski et al.,

⇑ Corresponding author. Tel.: +972 4 9539552; fax: +972 4 9836936. E-mail address: [email protected] (E. Lewinsohn). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.05.018

2010). Little is known about the biosynthesis of phenylpropylamino alkaloids in plants. Alkaloids are important plant metabolites belonging to diverse structural categories that are largely unrelated biosynthetically, in contrast to other groups of natural products such as the terpenoids and phenylpropanoids, which share common chemical structures and biosynthetic pathways (Croteau et al., 2000). Experimental evidence has indicated that the phenylpropylamino alkaloids are not derived via the simple decarboxylation involved in the biosynthesis of phenylethylamino alkaloids, such as tyramine. Feeding experiments have shown that exogenous L-pyruvate and L-phenylalanine is converted to ephedrine in Ephedra gerardiana stems (Grue-Sørensen and Spenser, 1988, 1994) via phenylacetylcarbinol, 1-phenylpropane-1,2-dione and (S)-cathinone intermediates (Fig. 1). The authors suggested that

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R. Krizevski et al. / Phytochemistry 81 (2012) 71–79 O OH NH2

H

L-Phenylalanine

PAL O OH

H

OH

CH3 O

t-Cinnamic acid

(R)-Phenylacetyl carbinol (7) H

HO

BCL

CH3 O

CO2 O

O

(S)-Phenylacetyl carbinol (9)

CH3 O

O CH3

Benzaldehyde (3) O H3C

H OH

O

Pyruvate (4)

1-Phenylpropane-1,2-dione (5)

OH

R

NH2

(R)-2-Hydroxypropiophenone (6) O

O

CH3

CH3 HO

H2N

H

(S)-2-Hydroxypropiophenone (8)

H

(S)-Cathinone (10)

R1 H

HO

R2 OH

H CH3

H2N

CH3 H2N

H

(1S,2S)-Norpseudoephedrine (11)

(1R,2S)-Norephedrine (12)

NMT2

NMT1 H

HO

H

OH

H CH3

HN

CH3 HN

H CH3

(1S,2S)-Pseudoephedrine (13)

(1R,2S)-Ephedrine (14)

NMT4

NMT3 HO

H

H

OH

CH3 N H3C

CH3 N

H CH3

(1S,2S)-N-Methylpseudoephedrine (15)

H CH3

H3C

H CH3

(1R,2S)-N-Methylephedrine (16)

Fig. 1. The proposed biosynthetic pathway to ephedrine alkaloids in Ephedra spp. The pathway is based on early radiolabeling experiments (Grue-Sørensen and Spenser, 1988, 1994) and the presence of putative precursors (Krizevski et al., 2010). Solid arrows represent biochemically resolved reactions including phenylalanine ammonia lyase (PAL) (Okada et al., 2008); (S)-cathinone reductases (R1, R2) and (1R,2S)-norephedrine N-methyltransferase (NMT2) (Krizevski et al., 2010) and benzaldehyde carboxyligase BCL (Fig. 3) while dashed arrows represent biochemically unconfirmed reactions. The biosynthetic steps from trans-cinnamic acid to benzaldehyde and the amine donor for the biosynthesis of (S)-cathinone are still unknown.

ephedrine alkaloid biosynthesis begins with a unique key skeleton forming reaction capable of condensing pyruvate with a ring-containing moiety such as benzaldehyde, benzoic acid or benzoyl CoA to form phenylacetylcarbinol or 1-phenylpropane-1,2-dione (Fig. 1). Following the formation of 1-phenylpropane-1,2-dione transamination postulatedly takes place producing (S)-cathinone, which is stereospecifically reduced and N-methylated to release

(1R,2S)-ephedrine and (1S,2S)-pseudoephedrine (Grue-Sørensen and Spenser, 1988, 1994). A similar pathway was earlier postulated by Leete (1958) to be operational in Catha edulis (Celastraceae), a plant that is chewed in the Middle East and East Africa for its stimulant properties (Krizevski et al., 2007). Recently, the presence of (S)-cathinone reductases and (1R,2S)-norephedrine N-methyltransferase enzymatic activities in cell-free extracts of Ephedra

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sinica stems and C. edulis leaves has been reported (Krizevski et al., 2007, 2010). Although Ephedra spp. are worldwidely distributed, it seems that the New world species lack ephedrine alkaloids. Within the Eurasian species, some Ephedra species such as E. fragilis and E. monosperma accumulate (1R,2S)-ephedrine, other species such as E. distachya and E. pachyclada accumulate only (1S,2S)-pseudoephedrine. Other Ephedra species such as E. foeminea, E. altissima and E. przewalskii completely lack both (1R,2S)-ephedrine and (1S,2S)-pseudoephedrine (Caveney et al., 2001). We have previously shown that Ephedra foeminea, an Eurasian species that lacks both (1R,2S)-ephedrine and (1S,2S)-pseudoephedrine also lacks other ephedrine alkaloids as well as their putative committed metabolic precursors 1-phenylpropane-1,2-dione and (S)-cathinone (Krizevski et al., 2010). The absence of 1-phenylpropane-1,2-dione in E. foeminea could be regarded as a metabolic bottleneck that might result from lack of enzymatic activity that forms 1-phenylpropane-1,2-dione or could be due to limitation of the assumed substrates pyruvate and either benzaldehyde, benzoic acid or benzoyl CoA for this reaction to take place. In this study we present evidence for the existence of a novel benzaldehyde carboxyligase enzymatic activity in cell-free extracts derived from Ephedra capable of decarboxylating pyruvate and ligating it to benzaldehyde to form (R)-phenylacetylcarbinol, (S)-phenylacetylcarbinol, (R)-2-hydroxypropiophenone, and (S)-2hydroxypropiophenone, in the presence of magnesium ions and thiamine pyrophosphate. We also report that the levels of this activity which probably catalyzes the first committed step towards ephedrine alkaloids, are in accordance with the tissue distribution patterns of ephedrine alkaloids in E. sinica and the lack of ephedrine alkaloids in E. foeminea. We also show that (S)-cathinone reductase might be also limiting ephedrine alkaloids biosynthesis in E. foeminea. Interestingly although the initial committed enzymes of the pathway are seemingly inactive in E. foeminea, a later downstream enzyme in the pathway, (1R,2S)-norephedrine N-methyltransferase is highly active.

2. Results A comparison of the volatiles present in E. sinica and E. foeminea by solid-phase microextraction (SPME) indicated that 1-phenylpropane-1,2-dione was only present in E. sinica and not in E. foeminea (Fig. 2A). Both plants also contained benzaldehyde, but E. foeminea (a species that lacks ephedrine alkaloids) displayed much higher levels of benzaldehyde (Fig. 2B), suggesting that benzaldehyde might be a precursor for 1-phenylpropane-1,2-dione and ephedrine alkaloids in Ephedra spp. In bacterial and yeast systems, pyruvate is readily decarboxylated and the resulting hydroxyethylthiamin diphosphate carbanion coupled to benzaldehyde to generate (R)-phenylacetylcarbinol, which is the commercial precursor to ephedrine (Meyer et al., 2011). We thus tested the possibility that cell-free extracts derived from E. sinica stems support such catalysis. Indeed, substantial levels of (R)-phenylacetylcarbinol, (S)phenylacetylcarbinol, as well as (R)-2-hydroxypropiophenone, (S)-2-hydroxypropiophenone and 1-phenylpropane-1,2-dione were generated in the presence of magnesium ions and thiamine pyrophosphate (TPP) by cell-free extracts of E. sinica (Fig. 3A). Enzymatic activity was detected in E. foeminea stems, but its levels were significantly lower than those detected in E. sinica cell-free extracts (Fig. 3B). Benzaldehyde carboxyligase (BCL) activity did not include benzoic acid or benzoyl CoA as substrates (Fig. 3C). In order to more accurately address differences in specific BCL activity between cell-free extracts derived from E. sinica or E. foeminea stems, BCL activity was measured at the linear range with constant pyruvate levels and varying benzaldehyde

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Fig. 2. SPME GC–MS analysis of volatiles from Ephedra spp. stems. (A) Young E. sinica stems. (B) Young E. foeminea stems. (C) authentic standards. Numbers were assigned according to the metabolic pathway shown in Fig. 1. (3) Benzaldehyde, (5) 1-Phenylpropane-1,2-dione. The experiment was repeated three times with similar results.

concentrations. At low benzaldehyde concentrations representing normal physiological conditions (7.5 mM) E. sinica BCL was 40 times faster than that of E. foeminea while at higher benzaldehyde concentrations (75 and 150 mM) E. sinica BCL was 10 times faster than that of E. foeminea (Fig. 4). Double reciprocal Lineweaver– Burke plot yielded apparent Vmax values of 1.3 and 0.15 (nmol/ min/mg) for E. sinica and E. foeminea as well as Km values of 17 and 100 mM, respectively. Thus, E. sinica BCL displayed an elevated maximum velocity and higher affinity towards benzaldehyde than that of E. foeminea. BCL activity in different E. sinica tissues was analyzed. Low activity levels were detected in stem tips with higher activity at middle stem sections and highest activity at stem base section with lowest levels in roots (Fig. 5), consistent with alkaloid levels (see below). In order to better understand additional biochemical factors that limit ephedrine alkaloid accumulation in E. foeminea, the levels of (S)-cathinone reductase enzymatic activity were compared between cell-free extracts derived from E. sinica and E. foeminea stems. The stereospecific reduction of (S)-cathinone in the presence of NADH to yield (1R,2S)-norephedrine and (1S,2S)-norpseudoephedrine has been previously described in E. sinica (Krizevski et al., 2010). As expected, E. sinica NADH dependent (S)-cathinone reductase enzymatic activity was noticeable, while E. foeminea had extremely low activity levels (Fig. 6). NADPH could not be utilized as a coenzyme in these reactions and heat inactivated proteins did not yield any of the biosynthetic products (Fig. 6C). Thus, two important reactions of the biosynthetic pathway to ephedrine alkaloids, namely benzaldehyde carboxyligase (Fig. 1, BCL) as well as a NADH (S)-cathinone reductase activity (Fig. 1, R1 and R2) seem limiting in E. foeminea. To evaluate if a downstream activity in the pathway also further limits the formation of ephedrine alkaloids in E. foeminea, the

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Fig. 3. Benzaldehyde carboxyligase enzymatic activity in young E. sinica and E. foeminea stems. Cell-free protein extracts were incubated with 75 mM benzaldehyde and 75 mM pyruvate in the presence of 5 mM magnesium chloride and 2 mM thiamine pyrophosphate. (A) Complete assay of an E. sinica extract. (B) Complete assay of an E. foeminea extract. (C) Complete assay with pyruvate and either benzoic acid or benzoyl CoA as co-substrates (other controls such as heat inactivated enzyme, complete assays lacking one or all substrates or cofactors of both plants, were devoid of activity, not shown). The enzymatic reaction was analyzed using GC–MS and the products 1-phenylpropane-1,2-dione (5), (R)-2-hydroxypropiophenone (6), (R)-Phenylacetyl carbinol (7), (S)-2-hydroxypropiophenone (8) and (S)phenylacetyl carbinol (9) were identified by comparison of their retention times and mass spectra to that of authentic standards. The experiment was repeated three times with similar results.

levels of (1R,2S)-norephedrine N-methyltransferase activity (Fig. 1, NMT2) were compared in E. sinica and E. foeminea cell-free extracts. This activity catalyzes the conversion of (1R,2S)-norephedrine to (1R,2S)-ephedrine in the presence of S-adenosylmethionine (SAM) as the methyl donor (Krizevski et al., 2010). NMT2 activity was readily detected in E. sinica cell-free extracts as expected (Fig. 7A). Surprisingly E. foeminea cell-free extracts also displayed (1R,2S)-norephedrine N-methyltransferase activity at significantly higher levels than those displayed by E. sinica extracts (Fig. 7B). The product (1R,2S)-ephedrine was not detected when a heat inactivated protein was used, nor in the absence of SAM (Fig. 7C) either in E. sinica nor E. foeminea cell-free extracts. 3. Discussion The intricate biochemical mechanisms that lead to the formation and accumulation of ephedrine alkaloids in Ephedra spp. and in other plants are largely unknown. In order to probe the biosynthetic pathway to ephedrine alkaloids we performed targeted metabolic profiling coupled to comparative biochemical analyses of

two Ephedra spp. that vary in their alkaloid content. E. sinica, a cultivated species that accumulates substantial levels of ephedrine alkaloids was compared to E. foeminea (syn. E. campylopoda), an Eurasian species that grows locally in Israel and does not accumulate ephedrine alkaloids (Caveney et al., 2001; Krizevski et al., 2010). Previous work enabled the identification of many pathway intermediates including 1-phenylpropane-1,2-dione and (S)-cathinone in E. sinica (Krizevski et al., 2010). E. foeminea lacks ephedrine alkaloids and also these precursors (Krizevski et al., 2010). Our present study indicated that benzaldehyde was present in the volatile fraction of E. sinica (Fig. 2A) as reported before (Tellez et al., 2004), but, E. foemina stems had much higher levels of benzaldehyde than E. sinica (Fig. 2B). Benzaldehyde together with pyruvate can act as substrates in bacterial acetohydroxyacid synthases to generate (R)-phenylacetylcarbinol and (S)-phenylacetylcarbinol (Stanislav et al., 2003; Chipman et al., 2005). In alcoholic fermentation in yeast and bacteria, pyruvate decarboxylases also catalyze the nonoxidative decarboxylation of pyruvate into acetaldehyde and CO2 requiring thiamine diphosphate and Mg2+ for catalytic activity. Microbial pyruvate decarboxylases cannot normally accept a second pyruvate molecule as a substrate to release (S)acetolactate, but also catalyze carboligation side reactions (Meyer et al., 2011). This property is exploited for the commercial asymmetric synthesis of (R)-phenylacetylcarbinol, that is chemically converted to (1R,2S)-ephedrine in commercial production (Stanislav et al., 2003; Meyer et al., 2011). We thus hypothesized that such BCL activities could be present in Ephedra tissues and might be involved in alkaloid biosynthesis. Moreover, it could be that benzaldehyde accumulated in E. foeminea due to its apparent inability to be further metabolized into alkaloids. In order to verify this hypothesis, we first determined if such a reaction was supported in cell-free extracts derived from E. sinica stems. The conversion of pyruvate and a second substrate that could possibly be either benzaldehyde, benzoic acid or benzoyl CoA into (R)-phenylacetylcarbinol, (S)-phenylacetylcarbinol, (R)2-hydroxypropiophenone and (S)-2-hydroxypropiophenone and 1-phenylpropane-1,2-dione clearly requires an initial step of pyruvate decarboxylation prior to its carboligation to the second substrate. Indeed, pyruvate and benzaldehyde were readily converted into the above oxygenated propyl benzene derivatives in E. sinica cell-free extracts, in the presence of thiamine pyrophosphate and magnesium ions (Fig. 3). This enzyme, benzaldehyde carboxyligase (BCL) readily accepted benzaldehyde but could not utilize benzoic acid or benzoyl CoA as substrates (Fig. 3C). Although benzoic acid was suggested to serve as a substrate for this reaction based on information derived from earlier precursor feeding experiments in E. gerardiana (Grue-Sørensen and Spenser, 1994), our data indicate that benzaldehyde is a preferred precursor for phenylpropylamino alkaloid biosynthesis as proposed in an earlier study by the same authors (Grue-Sørensen and Spenser, 1988). Both E. sinica and E. foeminea BCL are thiamine diphosphate dependent enzymes (Fig. 3). Most probably the Ephedra benzaldehyde carboxyligase proteins could be functionally related to the better studied prokaryotic acetolactate synthases and acetohydroxyacid synthases (Stanislav et al., 2003; Chipman et al., 2005; Duggleby et al., 2008), or to pyruvate decarboxylases. Acetolactate synthases produce acetolactate from two pyruvate molecules and are key enzymes in the biosynthesis of branched amino acids both in eukaryotic and prokaryotic organisms (Coruzzi and Last, 2000). Under normal physiological conditions acetolactate synthase decarboxylates one pyruvate molecule then carboligates it onto a second pyruvate molecule to release (S)-acetolactate, the first committed intermediate in the branched chain amino acids pathway. Intriguingly, both in prokaryotic and in yeasts, if benzaldehyde is offered as a substrate together with pyruvate, benzaldehyde carboxyligation takes place releasing 2 phenylacetylcarbinol isomers

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Fig. 4. Benzaldehyde carboxyligase specific activity at different benzaldehyde concentrations, activity was measured colorimetrically. The solid line represents cell-free extracts derived from E. sinica stem tips, the dashed line represents cellfree extracts derived from E. foeminea stem tips. The data presented are an average of four replicates ± s.e.

(Fig. 1, BCL) (Stanislav et al., 2003; Rosche et al., 2003; Chipman et al., 2005). Moreover, although differing in primary structure, bacterial pyruvate decarboxylases also catalyze the decarboxylation of pyruvate to acetaldehyde as part of the glycolysis cycle (Shrestha et al., 2010). Still, due to enzyme promiscuity, in addition to the decarboxylation of 2-keto acids, pyruvate decarboxylases catalyse the enantioselective formation of (R)-phenylacetylcarbinol, the industrial precursor of (1R,2S)-ephedrine and (1R,2S)-norephedrine (Meyer et al., 2011). Interestingly, mutated Zymomonas mobilis pyruvate decarboxylases give rise to (R)- and (S)-phenylacetylcarbinol, (S)-2-hydroxypropiophenone but not (R)-2hydroxypropiophenone (Pohl et al., 1998). It is possible that an ALS enzyme, rather than a PDC enzyme, catalyzes the key carboligation step in Ephedra spp. It is also possible that both enzyme types contribute to the observed BCL activity. Engel and colleagues have shown that ALS (more broadly called acetohydroxyacid synthase) from Escherichia coli efficiently catalyzes the synthesis of R-PAC from pyruvate and benzaldehyde, and can accept a broad range of benzaldehyde analogues as well (Engel et al., 2003, 2004). Although ALS homologues have been found in plants such as Arabidopsis (McCourt et al., 2006), activity with benzaldehyde substrate has not been shown. Our evidence indicates that cell-free extracts from young E. sinica and E. foeminea stems catalyze the formation of four phenylacetylcarbinol isomers as well as 1-phenylpropane-1,2-dione (Fig. 3). It could be that the later is formed by oxidation of phenylacetylcarbinol isomers by a yet unidentified oxidoreductase to generate the typical diketone functional group. Since the cell-free extracts utilized consisted of a mixture of enzymes we presently do not know how many distinct proteins are involved in these conversions. In any case, this is the first evidence that plant enzymes are capable of catalyzing benzaldehyde carboxyligation reactions. It is reasonable to assume that under normal physiological conditions BCL will encounter relatively low levels of benzaldehyde, in this case the BCL from E. foeminea will be 40 times less effective than that of E. sinica. The maximal velocity in which the reaction was catalyzed was much higher in E. sinica relative to E. foeminea and the affinity towards benzaldehyde as a substrate was significantly higher in E. sinica than that of

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E. foeminea. Moreover, it can be argued that E. foeminea BCL activity would be largely ineffective mostly because of its poor ability to compete for pyruvate which is a primary metabolite participating in many biochemical processes within a cell. For example, pyruvate decarboxylase efficiently converts pyruvate into acetaldehyde, pyruvate carboxylase releases oxaloacetate from pyruvate, ATP and CO2, pyruvate dehydrogenase forms acetyl CoA from pyruvate and lactate dehydrogenase converts pyruvate to lactic acid (Shrestha et al., 2010; Jitrapakdee and Wallace, 1999; Arai et al., 2001). This could partially explain the absence of the phenylcarbinol and hydroxypropiophenone isomers, 1-phenylpropane-1,2-dione and other downstream alkaloids in E. foeminea plants. Still, we cannot exclude the possibility that subcellular compartmentization could also play a role in the lack of ephedrine alkaloids accumulation in E. foeminea. Previous observations indicated that E. sinica plants accumulate low levels of ephedrine alkaloids in apical meristems, and the levels increase in lower parts of the stems, while roots lack ephedrine alkaloids (O’Dowd et al., 1998). The BCL specific activity distribution pattern (Fig. 5) correlates well with this previously determined ephedrine alkaloids distribution pattern in E. sinica. The low BCL specific activity levels detected in extracts from roots may not be of physiological relevance since its substrate benzaldehyde is absent from root tissues (data not shown). The next reaction taking place in the ephedrine alkaloids pathway following the formation of 1-phenylpropane-1,2-dione is its transamination to release (S)-cathinone (Fig. 1), but the amine donor as well as the biochemical conditions required for this reaction are still unknown. Nevertheless, the conversion of (S)-cathinone into (1R,2S)-norephedrine and (1R,2S)-norpseudoephedrine has been documented in E. sinica and C. edulis (Krizevski et al., 2007, 2010). Indeed, cell-free extracts derived from E. foeminea were devoid of (S)-cathinone reductase activity (Fig. 6), indicating that the pathway to alkaloid formation is further blocked at this biosynthetic step. Our data thus indicate that at least two reactions leading into the alkaloid pathway (namely BCL and (S)-cathinone reductase) are apparently limiting in E. foeminea (Figs. 3 and 6). This also further supports our previous data indicating that (S)-cathinone reductases are important enzymes controlling the stereospecificity of the alkaloids accumulated in E. sinica accessions and acting as a key factor in the determination of

Fig. 5. Benzaldehyde carboxyligase specific activity in different E. sinica tissues. The data presented are an average of five replicates ± s.e. The colorimetric assay was used.

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Fig. 6. Cathinone reductase activity in young E. sinica and E. foeminea stems. Cellfree protein extracts were incubated with (S)-cathinone and NADH. (A) Complete assay using E. sinica extracts. (B) Complete assay using E. foeminea extracts. (C) Heat inactivated enzyme (other controls such as complete assays lacking (S)-cathinone or NADH, were devoid of activity in both plants, not shown). The enzymatic reaction was analyzed using GC–MS and the products (1S,2S)-norpseudoephedrine (11) and (1R,2S)-norephedrine (12) were identified by comparison of their retention times and mass spectra to that of authentic (1R,2S)-norephedrine standard. The experiment was repeated three times with similar results.

ephedrine alkaloids composition and content (Krizevski et al., 2010). Unexpectedly, the N-methylation step that generates (1R,2S)ephedrine from (1R,2S)-norephedrine is active both in E. sinica and in E. foeminea (Fig. 7). Since norephedrine does not normally accumulate in E. foeminea tissues, one can only speculate on the actual biological role of this activity. It could be that the N-methyltransferase is a promiscuous enzyme exhibiting broad substrate specificity, enabling it to act on norephedrine, but its actual biological substrate is different and presently unknown. It could also be that the N-methyltransferase activity is an evolutionary relic trait that has not been fully selected against yet. Such latent or concealed pathways are often referred as ‘‘silent metabolic pathways’’ and they seem widely spread in many biosynthetic pathways in plant specialized metabolism (Lewinsohn and Gijzen, 2009).

Fig. 7. Norephedrine N-methyltransferase activity in young E. sinica and E. foeminea stems. Cell-free protein extracts were incubated with (1R,2S)-norephedrine and Sadenosylmethionine (SAM). (A) Complete assay using E. sinica cell-free extracts. (B) Complete assay using E. foeminea cell-free extracts. (C) Heat inactivated enzymes (other controls such as complete assays lacking (1R,2S)-norephedrine or SAM, were devoid of activity in both plants, not shown). The enzymatic reaction was analyzed by GC–MS and the product (1R,2S)-ephedrine (14) was identified by comparison of its retention time and mass spectrum to that of an authentic standard. The experiment was repeated three times with similar results.

Further work at the biochemical, metabolomic, transcriptomic and genomic levels is needed to better understand the biosynthetic pathways to ephedrine alkaloids in Ephedra and other species (Hagel et al., 2011, in press). Ephedrine alkaloids are restricted in the plant kingdom but their presence in taxonomically unrelated plants may reflect a case of convergent evolution in specialized metabolism (Pichersky and Lewinsohn, 2011).

4. Experimental 4.1. Plant material and chemical standards Ephedra foeminea Forssk. (syn. E. campylopoda C.A. Mey) stems were collected from wild populations growing in Kibbutz Yifat,

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and Alonei Abba, Jezreel Valley, Northern Israel. E. sinica seeds were purchased from Horizon Herbs, Williams, OR, USA. The seeds were originally collected from wild open pollinated E. sinica populations in northern China. Seeds were germinated and planted in 20 l pots containing a compost and tuff mixture. Ephedra plants were grown outdoors at the Newe Ya’ar Research Center in Northern Israel. The plants used in this study were 4 years old. Authentic standards of 1-phenyl-1,2-propanedione, (1R,2S)-norephedrine and (1R,2S)-ephedrine were purchased from Sigma Chem. Co., St. Louis, MO, USA. Benzaldehyde was purchased from Fluka AG Buchs, Switzerland. Pyruvate was purchased from Merck Chem. Co., Darmstadt, Germany. (S)-Cathinone was synthesized by oxidation of (1R,2S)-norephedrine using KMnO4 (Krizevski et al., 2007). (R)-2-Hydroxypropiophenone, (R)-phenylacetylcarbinol, (S)-2hydroxypropiophenone and (S)-phenylacetylcarbinol were gifts from Prof. David Chipman’s and Prof. Zeev Barak’s labs in Ben Gurion University. 4.2. Extraction of volatile metabolic precursors Freshly picked E. sinica or E. foeminea stems (1 g) were ground with a mortar and pestle in the presence of liquid nitrogen and placed in a 10 ml DuPont autosampler vial (DuPont-Dow Elastomers LLC, Wilmington, DE) with a white solid-top polypropylene cap (Alltech). Samples were overlaid with 3 ml NaCl (25%) solution and 1 g NaCl (for inhibition of enzyme activity). Samples were vigorously shaken for 30 s and incubated at room temperature for 1 h, and thereafter volatile compounds were collected with an SPME device PDMS-100 with a PDMS/DVB fiber (polymethylsiloxane/ divinylbenzene 65 lm, Supelco, Bellefonte, PA) by inserting the fiber into the tube and leaving it in place for 30 min at room temperature. After this incubation step, the SPME fiber was injected directly into the GC–MS apparatus. 4.3. Gas chromatography–mass spectrometry of volatile metabolic precursors SPME volatile analysis was performed on a GC-MSD system (Agilent, USA). The instrument was equipped with Rtx-5 SIL column (30 m length  0.25 mm i.d., 0.25 lm film thickness, stationary phase 95% dimethyl- and 5% diphenyl polysiloxane). Helium (1.3 ml/min) was used as a carrier gas with splitless injection. The injector temperature was 250 °C, and the detector temperature was 280 °C. The following conditions were used: initial temperature 45 °C for 1 min, followed by a ramp of 45–150 °C at a rate of 3 °C/min and a second ramp of 150–280 °C at a rate of 10 °C/min. A quadrupole mass detector with electron ionization at 70 eV was used to acquire the MS data in the range of 41–350 m/z. The identification of the volatiles was assigned by comparison of their retention times and spectral data with those of authentic standards. 4.4. Enzyme extractions Benzaldehyde carboxyligase was extracted as follows. Young freshly picked E. sinica or E. foeminea stems (20 g) were ground with a mortar and pestle under liquid nitrogen, with 2 g of analytical grade sea sand and 2 g polyvinylpolypyrrolidone (PVPP). The crushed powder was transferred to a 250 ml Erlenmeyer flask that was chilled on ice. Two hundred milliliters of buffer A composed of: 50 mM Bis–Tris propane, pH 7 containing 10% (v/v) glycerol, 1% (w/v) polyvinylpyrrolidone (PVP-40) and 5 mM dithiothreitol were added. Both cathinone reductases and norephedrine N-methyltransferase enzymes were extracted using the same procedure but with buffer B composed of: 50 mM Bis–Tris propane, pH 8.5 containing 10% (v/v) glycerol, 1% (w/v) polyvinylpyrrolidone (PVP-40) and 5 mM dithiothreitol (Krizevski et al., 2010). The

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sample was then vigorously shaken for 30 s and filtered through one layer of Miracloth (Calbiochem) and centrifuged at 15,000g for 20 min at 4 °C. The resulting crude soluble protein fraction was transferred to a clean ice-chilled Erlenmeyer flask and a final 30% saturation ammonium sulfate was added to the sample followed by stirring for 5 min and 1 h incubation at 4 °C. The sample was centrifuged at 15,000g for 20 min at 4 °C. The precipitated proteins and polyphenols were discarded and additional ammonium sulfate was added to reach a 60% saturation level followed by stirring for 5 min and 1 h incubation at 4 °C. The sample was centrifuged at 15,000g for 20 min at 4 °C. The precipitated crude protein fraction was dissolved in 5 ml buffer. The remaining soluble fraction was transferred to a new Erlenmeyer flask and ammonium sulfate was again added reaching a 90% ammonium sulfate saturation level followed by 5 min stirring and 1 h incubation at 4 °C. The sample was centrifuged at 15,000g for 20 min at 4 °C. The precipitated crude protein fraction was dissolved in 5 ml buffer. Each precipitated protein fraction was dissolved in 5 ml buffer and desalted by gel permeation chromatography on a BioGel P-6 column at a flow rate of 1 ml/min using the same extraction buffer. Fractions of 1 ml volume were evaluated for their protein content using the Bradford reagent (Bradford, 1976), with bovine serum albumin (BSA) as standard. The first 5 fractions showing the highest levels of protein were combined and used in further experiments. Heat inactivated enzyme was obtained by boiling the enzyme in 100 °C for 20 min. 4.5. Benzaldehyde carboxyligase enzymatic assay The enzyme assay reaction mixture for the decarboxylation of pyruvate and carboligation of the resulting acetaldehyde moiety to benzaldehyde consisted buffer A, 75 mM benzaldehyde (dissolved in 10% (w/v) Gum Arabic solution), 75 mM pyruvate, 2 mM thiamine pyrophosphate, 5 mM MgCl2, 60 mM KCl and up to 0.5 ml of protein extract (approximately 3.5 mg protein, 30– 60% ammonium sulfate fraction) in a total volume of 1 ml. After 1 h incubation at 37 °C, 4 ml of MTBE were added to the sample that was then vigorously shaken for 30 s and centrifuged at 10g for 5 min. The top organic fraction was collected and the aqueous residue was reextracted with 4 additional ml of MTBE. The pooled ethereal extracts were dried by the addition of anhydrous Na2SO4 and then evaporated under a gentle stream of N2 to a final volume of 0.25 ml. One microliter was injected into the GC–MS instrument for analysis. 4.6. Cathinone reductase enzymatic assays The enzyme assay reaction mixture for the reduction of (S)cathinone consisted buffer B, 5 mM (S)-cathinone, 5 mM NADH and up to 0.5 ml of protein extract (approximately 3.5 mg protein, 30–60% ammonium sulfate fraction) in a total volume of 1 ml. After 1 h incubation at 37 °C the reaction was stopped by the addition of 1 ml 1 N NaOH. The alkaloids were extracted twice with 4 ml MTBE. The ether sample was then evaporated and treated as above and analyzed by GC–MS. 4.7. Norephedrine N-methyltransferase enzymatic assays The enzyme assay reaction mixture for the methylation of (1R,2S)-norephedrine consisted in buffer B, 5 mM (1R,2S)-norephedrine, 5 mM SAM and up to 0.5 ml protein extract (approximately 2 mg protein, 60–90% ammonium sulfate fraction) in a total volume of 1 ml. After an over-night incubation at 37 °C the reaction was stopped by the addition of 1 ml 1 N NaOH. The alkaloids were extracted twice with 4 ml MTBE. The ether sample was then evaporated and treated as above and analyzed by GC–MS.

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4.8. Gas chromatography–mass spectrometry of enzymatic products

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The analysis was performed with a Hewlett–Packard GCD gas chromatograph equipped with a WCOT Restek Rt-bDEXsm fused silica column (30 m  0.25 mm  0.25 lm). The gas chromatograph was operated in splitless injector mode using 6 min delay time. Helium was used as the carrier gas with a flow rate of 0.8 ml/min. The injector and detector temperatures were 230 °C. The oven was set to the following temperature program: initial temperature 100 °C for 1 min, followed by a ramp of 100–165 °C at a rate of 1.5 °C/min and a second ramp of 165–220 °C at a rate of 10 °C/min. A quadrupole mass detector with electron ionization at 70 eV was used to acquire the MS data in the range of 41–350 m/z. The identification of the volatiles was assigned by comparison of their retention times and spectral data with those of authentic standards (Krizevski et al., 2008).

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4.9. Extraction of benzaldehyde carboxyligase for colorimetric assays Young freshly picked Ephedra stems (2 g) were ground with a mortar and pestle under liquid nitrogen, with 1 g of analytical grade sea sand and 1 g polyvinylpolypyrrolidone (PVPP). The crushed powder was transferred to a 20 ml Erlenmeyer flask that was chilled on ice with 8 ml of buffer A. The sample was then vigorously shaken for 30 s and centrifuged at 15,000g for 10 min at 4 °C and the supernatant was filtered through one layer of Miraclothinto a new ice chilled 20 ml Erlenmeyer flask. From this protein fraction 2.5 ml were desalted twice by gel permeation chromatography as depicted above. 4.10. Colorimetric determination of benzaldehyde carboxyligase enzymatic activity The enzyme assay reaction mixture for the decarboxylation of pyruvate and carboligation of the resulting acetaldehyde moiety to benzaldehyde consisted of buffer A, 75 mM benzaldehyde (dissolved in 10% Gum Arabic solution), 75 mM pyruvate, 2 mM thiamine pyrophosphate, 5 mM MgCl2, 60 mM KCl and up to 125 ll of protein extract (approximately 0.45 mg protein) in a total volume of 250 ll. After 3 h incubation at 37 °C the reaction was terminated by the addition of 25 ll H2SO4 followed by vigorous shaking for 30 s and reincubated at 37 °C for 30 min. The following solutions were added to the sample 450 ll DDW, 500 ll creatine (0.05 g in 10 ml DDW w/v) and 500 ll 1-naphthol (0.5 g in 10 ml 2.5 N NaOH w/v) and the sample was vigorously shaken for 30 s followed by reincubation at 37 °C for 15 min. Absorbance was measured at wavelengths of 490 nm and 580 nm for each sample, boiled enzyme control used for the calibration of spectrophotometer. Calculation was preformed according to calibration with authentic (R)-phenylacetylcarbinol standard as previously described (Stanislav et al., 2003). Linearity with time and protein content was established as part of the experimental setup. Acknowledgements This work was supported by the Israel Science Foundation (Grant No. 814/06 to E.L.) and a grant (CA-9117-09) from the Canada-Israel Binational Agricultural Research and Development Fund to E.L., F.M. and P.J.F. K. Kilpatrick is co-supervised by Norman P.A. Hüner (Dept. Biology, Western University).We thank Prof. Ze’ev Barak and Prof. David Chipman for fruitful discussions.

R. Krizevski et al. / Phytochemistry 81 (2012) 71–79 Stanislav, E., Vyazmensky, M., Geresh, S., Barak, Z., Chipman, D.M., 2003. Acetohydroxyacid synthase: A new enzyme for chiral synthesis of Rphenylacetylcarbinol. Biotechnol. Bioeng. 83, 833–840.

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Tellez, M.R., Khan, I.A., Schaneberg, B.T., Crockett, S.L., Rimando, A.M., Kobaisy, M., 2004. Steam distillation–solid-phase microextraction for the detection of Ephedra sinica in herbal preparations. J. Chromatogr. A 1025, 51–56.