Evolution of Morphine Biosynthesis in Opium Poppy

Home , Codeinone, Magnoflorine, Papaver arenarium, Papaver bracteatum, Papaver commutatum, Papaver glaucum

Phytochemistry 70 (2009) 1696–1707

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Phytochemistry

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Review Evolution of morphine biosynthesis in opium poppy

Jörg Ziegler a,b, Peter J. Facchini b, René Geißler a,1, Jürgen Schmidt a, Christian Ammer a, Robert Kramell a, Susan Voigtländer a, Andreas Gesell a,2, Silke Pienkny a, Wolfgang Brandt a,* a Leibniz-Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany b Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4 article info abstract

Article history: Benzylisoquinoline alkaloids (BIAs) are a group of nitrogen-containing plant secondary metabolites com- Received 7 May 2009 prised of an estimated 2500 identified structures. In BIA metabolism, (S)-reticuline is a key branch-point Received in revised form 29 June 2009 intermediate that can be directed into several alkaloid subtypes with different structural skeleton config- Available online 6 August 2009 urations. The morphinan alkaloids are one subclass of BIAs produced in only a few plant species, most notably and abundantly in the opium poppy (Papaver somniferum). Comparative transcriptome analysis Keywords: of opium poppy and several other Papaver species that do not accumulate morphinan alkaloids showed Papaver somniferum that known genes encoding BIA biosynthetic enzymes are expressed at higher levels in P. somniferum. Papaveraceae Three unknown cDNAs that are co-ordinately expressed with several BIA biosynthetic genes were iden- Morphine Benzylisoquinoline alkaloids tified as enzymes in the pathway. One of these enzymes, salutaridine reductase (SalR), which is specific Transcript profiling for the production of morphinan alkaloids, was isolated and heterologously overexpressed in its active Metabolite profiling form not only from P. somniferum, but also from Papaver species that do not produce morphinan alkaloids. Short chain dehydrogenase/reductase SalR is a member of a class of short chain dehydrogenase/reductases (SDRs) that are active as monomers and possess an extended amino acid sequence compared with classical SDRs. Homology modelling and substrate docking revealed the substrate binding site for SalR. The amino acids residues conferring salutaridine binding were compared to several members of the SDR family from different plant species, which non-specifically reduce ()-menthone to (+)-neomenthol. Previously, it was shown that some of these proteins are involved in plant defence. The recruitment of specific monomeric SDRs from monomeric SDRs involved in plant defence is discussed. Ó 2009 Elsevier Ltd. All rights reserved.

Contents

1. Introduction ...... 1696 2. Integration of alkaloid and transcript profiles in Papaver species ...... 1699 3. Characterization of cDNAs involved in BIA metabolism based on transcriptional profiles ...... 1703 4. Occurrence of SalR in other Papaver species ...... 1703 5. Structure-function analysis of SalR ...... 1703 6. Relationship of SalR to other SDRs ...... 1705 Acknowledgments ...... 1706 References ...... 1706

1. Introduction widespread occurrence, mainly in the plant kingdom. Nine hun- dred out of 1000 plant species in which benzylisoquinoline alka- Benzylisoquinoline alkaloids (BIAs) are a structurally diverse loids (BIAs) have been detected belong to the closely related group of nitrogen-containing plant secondary metabolites with orders of the Ranunculales, Magniolales, and Laurales, but many structures have also been found in several species of the Rutaceae * Corresponding author. Tel.: +49 345 5582 1360; fax: +49 345 5582 1309. (Shulgin and Perry, 2002). Thus, the occurrence of this class of E-mail address: [email protected] (W. Brandt). plant secondary metabolites across several plant families might re- 1 Present address: Institute of Biochemistry and Biotechnology, Martin-Luther- ﬂect both monophyletic origins and parallel evolutionary events University Halle-Wittenberg, D-06120 Halle, Germany. 2 Present address: Department of Biology, University of Victoria, Victoria, BC, (Jensen, 1995). The evolution and diversiﬁcation of metabolic path- Canada V8 W 3N5. ways is believed to result from the recruitment of enzymes from

0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.07.006 J. Ziegler et al. / Phytochemistry 70 (2009) 1696–1707 1697 primary metabolism. After gene duplication, random mutations in specific accumulation of distinct metabolites that are all produced new genes result in the establishment of novel catalytic functions via one pathway. (Ober and Hartmann, 2000). This mechanism was demonstrated BIA biosynthesis begins with a condensation reaction catalyzed for an enzyme in pyrrolizidine alkaloid biosynthesis. Homospermi- by norcoclaurine synthase of dopamine and para-hydroxyphenyl- dine synthase, the first enzyme in the pathway, was identified as a acetaldehyde, both of which are derived from L-tyrosine (Fig. 1). modified version of the ubiquitous enzyme deoxyhyposine syn- Interestingly, two enzymes belonging to two different protein fam- thase, which is responsible for translational regulation (Ober and ilies have been identified to catalyze the reaction. One enzyme be- Hartmann, 1999). Similarly, high sequence similarities suggest that longs to the PR10/Betv1 family of proteins and its recruitment from putrescine N-methyltransferase, which is the first committed step defence proteins is discussed (Samanani et al., 2004; Liscombe in tropane alkaloid metabolism, is derived from spermidine syn- et al., 2005). The other enzyme was classified as a member of the thase (Hashimoto et al., 1998; Teuber et al., 2007). In the same 2-oxoglutarate-dependent dioxygenase family, although it did pathway, the classification of tropinone reductases as members not possess an oxoglutarate binding site, and was active in the ab- of the short chain dehydrogenase/reductase (SDR) family suggests sence oxoglutarate (Minami et al., 2007). (S)-Norcoclaurine repre- the recruitment of these enzymes from primary metabolism (Nak- sents the first pathway intermediate with a benzylisoquinoline ajima et al., 1993). The emergence of these enzymes is characteris- skeleton from which all other BIA classes are derived. In a first ser- tic for another evolutionary process, in which new functions are ies of reactions, (S)-norcoclaurine is methylated by norcoclaurine established by small variations in existing functions. The modifica- 6-O-methyltransferase (6OMT) to (S)-coclaurine, which is N- tion of tropinone reductases led to the generation of two paralo- methylated by coclaurine N-methyltransferase (CNMT) yielding gous enzymes with opposite stereo-specificities (Nakajima et al., N-methylcoclaurine (Morishige et al., 2000; Choi et al., 2002; Oun- 1993). The two products serve as the precursors of two different aroon et al., 2003). Subsequent hydroxylation of the benzyl moiety classes of tropane alkaloids, scopolamine/hyoscyamine and the by (S)-N-methylcoclaurine 30-hydroxylase, a P450-dependent calystegines. A recently identified SDR from Cochlearia officinalis monooxygenase belonging to the CYP80B subfamily, and methyla- was suggested to represent a prototype during the development tion by (S)-30-hydroxy N-methylcoclaurine 40-O-methyltranferase of stereospecific tropinone reductase, since it forms a mixture of (4’OMT) results in the formation of (S)-reticuline (Pauli and Kut- tropine and pseudotropine from tropinone (Brock et al., 2008). chan, 1998; Huang and Kutchan, 2000; Morishige et al., 2000; Zie- The functional diversification and specialization of enzymes after gler et al., 2005). This basic benzylisoquinoline pathway is involved recruitment from common ancestors is also evident within the in the formation of most BIAs with the exception of the bisbenzyl- numerous plant secondary metabolic enzymes, which belong to isoquinolines and (most likely) the N-demethylated benzylisoquin- large protein families including chalcone synthases, terpenoid syn- olines, which are derived from precursors upstream of thases, O-orN-methyltransferases, glycosyltransferases, and P450- (S)-reticuline (Kraus and Kutchan, 1995; Pienkny et al., 2009). For dependent monooxygenases (Durbin et al., 2000; Vogt and Jones, all other BIAs, (S)-reticuline represents a major branch-point 2000; Zubieta et al., 2001, 2002; Austin and Noel, 2003; Ounaroon intermediate in the pathway. Methylations lead to the further et al., 2003; Schuler and Werck-Reichhart, 2003; Uefuji et al., 2003; elaboration of the simple benzylisoquinoline class with respect to Ziegler et al., 2005; Stenzel et al., 2006; Tholl, 2006; Inui et al., the number and positions of the methyl groups. The reactions 2007; Liscombe and Facchini, 2007). resulting in the majority of BIA structural diversity involve the for- Another strategy for the establishment of specific metabolite mation of C–C bonds between the isoquinoline ring, or the N- profiles involves molecular mechanisms that lead to the activa- methyl group, and the benzyl moiety. Depending on the positions tion or deactivation of genes. Cell- or tissue-specific accumulation of the carbon atoms, these reactions produce different classes of of plant secondary metabolites generally correlates with the BIAs, such as the pavines, isopavines, aporphines, proaporphines, expression of the corresponding biosynthetic genes, as shown and protoberberines (Shulgin and Perry, 2002). The latter group for tropinone reductases and phenylpropene O-methyltransfer- of compounds can be further modified to benzophenanthridines, ases (Nakajima and Hashimoto, 1999; Gang et al., 2002). Diver- rhoeadines, papaverrubines and phthalideisoquinolines (Fig. 1). gent metabolite profiles in individual plants, such as for the Subsequent reactions lead to further structural diversity within cultivar-specific accumulation of phenylpropenes in sweet basil each BIA class and include O- and N-methylations, formation of lines or the ecotype specific occurrence of distinct glucosinolates methylenedioxy bridges, oxidations, reductions, and hydroxyl- in Arabidopsis, could be related to differential gene expression ations. Whereas all these structural classes are derived from (S)- profiles (Gang et al., 2002; Lambrix et al., 2001; Hansen et al., reticuline, the promorphinan and morphinan alkaloids are derived 2008). from (R)-reticuline (Gerardy and Zenk, 1993a). Oxidation and With about 2500 known structures, BIA biosynthesis represents reduction by 1,2-dehydroreticuline synthase and 1,2-dehydroreti- a rich background for investigations on the mechanism leading to culine reductase, respectively, leads to the inversion of stereo- metabolic diversity in plants. BIA diversity results from modifica- chemistry (De-Eknamkul and Zenk, 1992; Hirata et al., 2004). tion of a basic carbon skeleton consisting of an isoquinoline and Subsequent C–C phenol coupling catalyzed by the P450-dependent a benzyl moiety (Ziegler and Facchini, 2008). This backbone struc- enzyme salutaridine synthase between C20 and C4a results in salu- ture is modified by a multitude of enzymes catalyzing various taridine (Gerardy and Zenk, 1993a; Gesell et al., 2009). Stereospe- types of reactions, such as hydroxylations, reductions, oxidations, cific reduction to 7(S)-salutaridinol by salutaridine reductase and C–C bond formations, and O- and N-methylations (Fig. 1). acetylation of the hydroxyl group by salutaridinol 7-O-acetyltrans- Accordingly, these enzymes belong to a variety of protein families ferase (SalAT) leads to salutaridinol 7-O-acetate (Gerardy and Zenk, including P450-dependent monooxygenases, short chain dehydro- 1993b; Lenz and Zenk, 1995a; Grothe et al., 2001; Ziegler et al., genases/reductases, flavin-dependent oxygenases, and O- and 2006). Thebaine can be formed from this compound either by N-methyltransferases. Although highly related to each other, the spontaneous rearrangement, or enzymatically by thebaine syn- individual members of these protein families show discrete sub- thase (Lenz and Zenk, 1994; Fisinger et al., 2006). Subsequent strate-, regio- and stereo-specificities, serving as ideal subjects to demethylations and one reduction by codeinone reductase investigate the evolution of enzyme function. Furthermore, BIA (CoR1) represent the final steps in morphine biosynthesis (Lenz profiles are species specific, and large variations can occur between and Zenk, 1995b; Unterlinner et al., 1999). even closely related plant species (Shulgin and Perry, 2002). This Cognate cDNAs encoding all enzymes of the basic pathway provides a tool to analyze the mechanism leading to the species- leading to (S)-reticuline have been cloned from a limited number 1698 J. Ziegler et al. / Phytochemistry 70 (2009) 1696–1707

Fig. 1. Benzylisoquinoline alkaloid biosynthesis. Multiple arrowheads denote more than one enzymatic step. Arrows without labelling reflect conversions that have not been enzymatically characterized. Enzymes that have been characterized are labelled, and those for which cognate cDNAs have been isolated are circled. The basic benzylisoquinoline pathway is shaded in dark grey, whereas the promorphinan and morphinan pathways are shaded in light grey. Abbreviations: 4-HPAA, para-hydroxyphenyl acetaldehyde; 40OMT, (S)30-hydroxy N-methylcoclaurine 40-O-methyltransferase; 6OMT, (S)-norcoclaurine 6-O-methyltransferase; 7OMT, (R,S)- reticuline 7-O-methyltransferase; BBE, berberine bridge enzyme; BBS, berbamunine synthase; CAS, (S)-canadine synthase; CFS, (S)-cheilanthifoline synthase; CNMT, (S)- coclaurine N-methyltransferase; CoOMT, columbamine O-methyltransferase; CoR1, codeinone reductase 1; CTS, (S)-corytuberine synthase (CYP80G2); DBOX, dihydrobenz- ophenanthridine oxidase; DRR, 1,2-dehydroreticuline reductase; DRS, 1,2-dehydroreticuline synthase; MSH, methylstylopine 14-hydroxylase; N7OMT, (S)-norreticuline 7-O-methyltransferase, NCS, (S)-norcoclaurine synthase; NMCH, (S)-N-methylcoclaurine 30-hydroxylase (CYP80B subfamily); P6H, protopine 6-hydroxylase; SalAT, 7(S)- salutaridinol 7-O-acetyltransferase; SalR, salutaridine reductase; SalSyn, salutaridine synthase (CYP719B1); SOMT, scoulerine 9-O-methyltransferase; STOX, (S)-tetrahydroprotoberberine oxidase; StyS, (S)-stylopine synthase; THS, thebaine synthase; TNMT; tetrahydroprotoberberine N-methyltransferase; TYDC, tyrosine decarboxylase. J. Ziegler et al. / Phytochemistry 70 (2009) 1696–1707 1699 of different species. However, cDNAs are available for only some of morphinan alkaloids. Such [M + H]+ ions are m/z 284.128669 (ele- + the enzymes involved in downstream pathways including the pro- mental composition [C17H18NO3] ) for morphinone, m/z + toberberine pathway enzymes berberine bridge enzyme (BBE), 286.144319 ([C17H20NO3] ) for morphine, m/z 298.144319 + scoulerine 9-O-methyltransferase, columbamine O-methyltrans- ([C18H20NO3] ) for oripavine, codeinone, and neopinone, m/z + ferase, canadine synthase, stylopine synthase, cheilanthifoline syn- 300.170533 ([C18H22NO3] ) for codeine, and m/z 312.159969 + thase and tetrahydroprotoberberine N-methyltransferase (Dittrich ([C19H22NO3] ) for thebaine, which represent all morphinan struc- and Kutchan, 1991; Takeshita et al., 1995; Morishige et al., 2002; tures (Fig. 2). Among 19 masses for promorphinan alkaloids repre- Ikezawa et al., 2003, 2007, 2009; Liscombe and Facchini, 2007; Zie- senting 28 structures, only two [M + H]+ ions are indicative for gler and Facchini, 2008). Cognate cDNAs have been reported for the intermediates in morphine biosynthesis. These are m/z + C–C phenol coupling enzyme CYP80G2 leading to aporphine alka- 328.154883 ([C19H22NO4] ) for salutaridine and m/z 330.170533 + loids (Ikezawa et al., 2008), and for reticuline 7-O-methyltransfer- ([C19H24NO4] ) for salutaridinol (Fig. 2). ase (7OMT), which leads to laudanine (Ounaroon et al., 2003; Fujii For analysis of the ESI-FTICR-MS data, only even-numbered et al., 2007). In the morphinan pathway, cDNAs have been identi- [M + H]+ masses larger than m/z 200, indicating the presence of fied for SalAT, SalR and CoR1 (Unterlinner et al., 1999; Grothe et an odd number of nitrogen atoms, were considered. This filter ex- al., 2001; Ziegler et al., 2006). Considering the remarkable struc- cludes the majority of small isoquinoline alkaloids, as well as bisb- tural diversity of BIAs, many more interesting biosynthetic en- enzylisoquinolines containing two nitrogens. The detected masses zymes and genes still await characterization. were assigned to a BIA mass table (mass window 0.0025), contain- In the past years, investigations aimed at the elucidation of the ing masses and empirical formulas. A visualization of the mass dis- evolution of the structural diversity resulting from BIA metabo- tribution across 15 Papaver species and six P. somniferum varieties lism. Initially, transcriptome and alkaloid profiles across different is shown in Fig. 3. related plant species were established as a first step to identify The most widely distributed [M + H]+ mass was at m/z 328 + gene expression patterns that could be related to the accumulation ([C19H22NO4] ), which was detected in seven species and four P. of specific BIAs. Subsequently, these transcript profiles were used somniferum varieties. This was not surprising considering that this to select candidate cDNAs for functional characterization with re- mass is representative of more than 70 different BIAs from almost spect to BIA metabolism. The enzymes encoded by these cDNAs every class (Shulgin and Perry, 2002). However, LC-MS/MS analysis were then compared to related enzymes involved in other plant identified this mass in the P. somniferum varieties Paso, Nopa, Fool- secondary metabolic pathways to investigate their evolutionary Ori and top1 as scoulerine and an aporphine-type alkaloid. The vari- origin. Subsequent structure-function analysis was performed to ety Paso also contained thebaine N-oxide. In Papaver bracteatum, identify decisive amino acids that might be responsible for specific this mass was identified as corytuberine and the morphinan alka- functions. loid thebaine N-oxide (Fig. 2). The occurrence of typical morphinan or promorphinan alkaloid fragments could not be observed in other 2. Integration of alkaloid and transcript profiles in Papaver samples. The elemental composition indicative for salutaridinol species ([M + H]+ at m/z 330), could only be detected in P. somniferum varieties, but was identified as reticuline by LC-MS/MS. Initially, there was the intention to identify transcriptional pro- Thebaine, the first morphinan alkaloid in the pathway, was de- files potentially correlated with the accumulation of a distinct class tected in five out of six P. somniferum varieties, and in P. bractea- of BIAs. The focus were the morphinan alkaloids due to their phar- tum. The corresponding mass ([M + H]+ m/z 312) in Papaver macological properties and extensive knowledge concerning their orientale and Papaver arenarium could not be identified as a mor- biosynthesis. Furthermore, the accumulation of morphinan alka- phinan or promorphinan alkaloid, but rather as the aporphine alkaloids is almost exclusively restricted to the opium poppy (Papaver loid isothebaine in P. orientale. The mass [M + H]+ m/z 298 being somniferum). Since the accumulation of these compounds is not in- indicative of about 20 BIAs was detected in the P. somniferum vari- duced by external stimuli in either intact plants or cultured cells, ety top1 as well as in Papaver fugax and Papaver pyrenaicum. the gene expression profiles of P. somniferum with several morphi- Among three morphinan alkaloids with this mass – neopinone, nan alkaloid-free Papaver species were compared. A potential codeinone, and oripavine – none of the three compounds could caveat of comparing the transcript profiles of different Papaver spe- be confirmed by LC-MS/MS. With the exception of top1, which cies was the high probability of differential gene expression caused did not show a signal at m/z 300 ([M + H]+), codeine was detected by (1) inconsistent developmental conditions despite harvesting in five out of six P. somniferum varieties. This mass was present in each plant at a similar growth stage, and (2) a lack of equivalent four other Papaver species, but could not be identified as morphi- morphology between species. In order to reduce the interference nan or promorphinan alkaloids. The occurrence of morphine in of plant development processes, such as growth, senescence, tissue the P. somniferum varieties was identical to codeine. The lack of co- differentiation, reproduction and seed maturation, stems were se- deine and morphine in top1 is consistent with the selection of this lected over leaves or seed capsules as the source for RNA. Stems P. somniferum mutant for this trait (Millgate et al., 2004). While the also possess a high capacity for BIA biosynthesis. The effect of po- [M + H]+ mass signal at m/z 286 was identified as morphine in five tential differences in stem morphology between Papaver species out of six P. somniferum varieties, in P. arenarium this ion was iden- was minimized by the inclusion of many species in the study. tified as N-demethylcodeine (Fig. 2). The number of differentially expressed genes was expected to de- Remarkably, none of the early pathway intermediates leading to cline when many species with varying morphologies were com- reticuline were detected in any of the plants suggesting a low pared, increasing the likelihood that cDNAs relevant to morphine abundance and rapid turnover of these compounds. Moreover, a biosynthesis were revealed. mass at m/z 284 indicative of morphinone was not detected. Mor- The alkaloid composition of various Papaver species is depen- phinone is a putative intermediate in one branch of the bifurcating dent on growth conditions, tissue source, and geographical origin. pathway from thebaine to morphine (Fig. 1). This pathway pro- Electrospray Fourier transform-ion cyclotron resonance mass spec- ceeds via oripavine, whereas codeine is an intermediate in the trometry (ESI-FTICR-MS) analysis was performed to establish the alternate branch. Only codeine, but not morphinone or oripavine, putative alkaloid composition for 15 Papaver species (Ziegler was detected in P. somniferum varieties containing morphine, et al., 2006). This pre-screening approach revealed the occurrence which suggests the involvement of only one of the branch path- of potentially diagnostic masses indicative of morphinan or pro- ways in these plants. 1700 J. Ziegler et al. / Phytochemistry 70 (2009) 1696–1707

Fig. 2. Masses ([M + H]+, m/z), elemental compositions, and structures of benzylisoquinoline compounds. Benzylisoquinoline alkaloids which have been detected and identiﬁed in Papaver species described in this study are shown in bold letters.

In summary, several morphinan and promorphinan alkaloids 2006). Hierarchical clustering of the transcript abundance data were only detected in P. somniferum varieties. Only one morphinan showed four clusters of ESTs displaying higher expression in P. and a corresponding derivative were found in P. bracteatum, which somniferum varieties, and three clusters of ESTs displaying higher accumulates high levels of thebaine. Surprisingly, the morphinan expression in other Papaver species (Fig. 4). A greater abundance derivative N-demethylcodeine was present in P. arenarium. This of transcripts in other Papaver species compared with P. somnife- species was previously not known to contain such alkaloids. rum suggests that differences in signal intensities were not caused Although its biosynthesis has not been investigated, it is reason- by low sequence homology between the P. somniferum ESTs and able to assume that N-demethylcodeine is derived from codeine the cDNAs from the other species. by N-demethylation. However, an alternative pathway involving In total, 102 ESTs were differentially expressed, 66 of which N-demethylated intermediates, perhaps starting from norreticu- showed higher expression levels in P. somniferum. These ESTs are line, cannot be ruled out. all differentially expressed in morphinan and promorphinan alka- Corresponding transcript profiles were produced by performing loid-accumulating and in morphinan-free Papaver species, irre- macroarray analysis on 54 individual plants belonging to the vari- spective of species morphology. Therefore, they may be regarded ous Papaver species used in this study. The macroarrays consisted as P. somniferum-specific transcripts and, thus, as putative morphi- of about 2000 unigenes obtained from two EST libraries prepared nan alkaloid-specific ESTs. Although not all of these ESTs are likely from P. somniferum stems and seedlings (Ziegler et al., 2005, involved in BIA metabolism, it is noteworthy that only a few were J. Ziegler et al. / Phytochemistry 70 (2009) 1696–1707 1701

Fig. 3. Visualization of ESI-FTICR-MS screening of Papaver species. The masses of the ESI-FTICR-MS analysis for each species were assigned to masses and empirical formulas for which BIAs exist. The presence of a mass is indicated by a grey bar. Closed arrows point to masses of promorphinan and morphinan alkaloids, open arrows point to other masses described in the text.

similar to proteins involved in general cellular function. The major- in the P. somniferum varieties Paso, Papa, and FoolOri (Pienkny ity (65% and 52% with higher and lower expression levels in P. som- et al., 2009). niferum, respectively) were not homologous to known proteins or BBE transcript levels were high in all P. somniferum lines except were similar to proteins of unknown function. the variety Paso. Higher BBE transcript levels were also found in All cDNAs encoding BIA biosynthetic enzymes showed higher Papaver glaucum, P. arenarium, Papaver commutatum, Papaver pavo- expression levels in P. somniferum plants. The abundance of tran- nium and P. pyrenaicum. BBE catalyzes the formation of a C–C bond scripts encoding for tyrosine decarboxylase, 6OMT, CYP80B3 and between the N-methyl group and the 20 position of the benzyl moi- 40OMT is remarkable since all belong to the basic benzylisoquin- ety of (S)-reticuline leading to (S)-scoulerine, which represents the oline pathway leading to (S)-reticuline and were expected to initial reaction leading to several BIA classes including the protob- occur at similar levels in the other Papaver species. Only in P. erberines and phthalideisoquinolines. A strong mass signal at 0 + + bracteatum and P. arenarium CYP80B3, 6OMT and 4 OMT were [M + H] m/z 414 ([C22H24NO7] ) was detected in the P. somniferum also expressed at higher levels compared to all other Papaver variety Nosca and at weaker levels in P. somniferum var. Paso and species. Additionally, all genes encoding enzymes acting down- FoolOri, and in P. glaucum (Fig. 3). Among known BIAs, this mass stream of (S)-reticuline were expressed at a higher level in P. is only representative of the phthalideisoquinoline alkaloid nosca- somniferum varieties. pine (Fig. 2), and it could be identified in Paso, FoolOri, and Nosca The coordinated expression of BIA biosynthetic genes has also by LC-MS/MS. The abundance of BBE transcripts in FoolOri and been reported for elicited P. somniferum cell cultures (Zulak et al., Nosca might reflect its involvement in noscapine biosynthesis. 2007). The high expression level of genes encoding SalSyn, SalR, Alternatively, BBE expression was low in Paso, which also appears SalAT, and CoR1 corroborates the abundance of morphinan and to accumulate noscapine, arguing against a direct link between BBE promorphinan alkaloids in P. somniferum. Transcripts for these en- transcript levels and the occurrence of noscapine. However, BBE is zymes are also abundant in P. arenarium and P. bracteatum, involved in the biosynthesis of more than a quarter of all known although CoR1 expression is lower in P. bracteatum. BIAs, suggesting that BBE expression might be related to the pro- Transcript levels for 7OMT were high in all P. somniferum duction of other alkaloids not identified in our analysis. It is varieties and in a few individuals from P. orientale, P. bracteatum remarkable that BBE transcript levels are lower in other Papaver and P. arenarium. This enzyme converts reticuline to laudanidine species. Although the content of BBE-derived alkaloids was not in the biosynthetic pathway to laudanosine (Ounaroon et al., investigated, some Papaver species accumulate protoberberines 2003). Masses indicative of both compounds ([M + H]+ m/z 344 as major alkaloids and higher BBE expression might have been ex- + + + [C20H26NO4] for laudanidine, [M + H] m/z 358 [C21H28NO4] pected (Shulgin and Perry, 2002). for laudanosine) were detected in all species that showed higher All BIA biosynthetic genes appear to show higher expression 7OMT gene expression (Fig. 3). LC-MS/MS analysis revealed the levels in P. somniferum compared with other Papaver species. In presence of both compounds in all P. somniferum varieties, as the case of genes encoding enzymes involved in the formation of well as the occurrence of laudanidine in P. orientale (Fig. 2). A morphinan and promorphinan alkaloids, the high expression levels second 7OMT also exhibiting higher transcript abundance in P. can be correlated to the unique alkaloid content of P. somniferum. somniferum, but specific for norreticuline, might be related to In contrast, this correlation is more unclear for enzymes such as + + the occurrence of papaverine ([M + H] m/z 340 [C20H22NO4] ) BBE or those acting upstream of (S)-reticuline. Potentially, the 1702 J. Ziegler et al. / Phytochemistry 70 (2009) 1696–1707

Fig. 4. Gene expression analysis in Papaver species. Seven clusters consisting of ESTs exhibiting increased (cluster 1–4) and decreased (cluster 5–7) expression in P. somniferum varieties are shown. Previously characterized cDNAs coding for enzymes in benzylisoquinoline alkaloid biosynthesis are indicated in blue, and cDNAs selected for characterization based on species-dependent expression proﬁles are shown in red. Only ESTs exhibiting signiﬁcant homologies to database entries are labelled. Each column of plant species represent individual plants.

extensive breeding of P. somniferum for high alkaloid accumulation thetic enzymes are still unknown, the tools to test this hypothesis might account for the phenomenon. Varieties with higher morphi- are presently not available. nan alkaloid content might be associated with the silencing of A high proportion of genes exhibiting differential expression genes leading to other BIA classes. However, since many biosyn- encode P450-dependent monooxygenases (Fig. 4). Transcripts for J. Ziegler et al. / Phytochemistry 70 (2009) 1696–1707 1703

five such cDNAs were more abundant in P. somniferum varieties, dine 7-O-acetyltransferase (Lenz and Zenk, 1995a; Grothe et al., whereas 9 occurred at lower levels. CYP80B3 and CYP719B1 were 2001). in the cluster containing other BIA biosynthetic genes (Fig. 4). Only The characterization of three novel cDNAs shows the potential members of these two P450 families have been implicated in BIA for gene discovery based on differential expression between P. metabolism, catalyzing ring hydroxylations, C–C and C–O phenol somniferum and other Papaver species. It will be interesting to coupling, and formation of methylenedioxy bridges (Kraus and see how many other genes displaying higher transcript levels in Kutchan, 1995; Pauli and Kutchan, 1998; Ikezawa et al., 2003, P. somniferum are involved in BIA biosynthesis, and whether some 2007, 2008, 2009). CYP80B3 is involved in the early pathway up- of the genes showing higher expression in other Papaver species stream of (S)-reticuline, and CYP719B1 has been characterized as are involved in BIA pathways specific to these plants. salutaridine synthase (Gesell et al., 2009). One P450 enzyme showed high similarity to the CYP73A subfamily, which includes 4. Occurrence of SalR in other Papaver species cinnamate 4-hydroxylase. The other P450 enzymes could not be unequivocally classified. Based only on the sequence similarity of Transcript profiling revealed high levels of SalR expression in all partial clones, no putative functions could be assigned to most of P. somniferum varieties, and moderate expression levels in P. the P450s exhibiting higher transcript levels in the other Papaver bracteatum and P. arenarium. Morphinan alkaloids were detected species. However, some can generally be assigned to the CYP71, in all of these species. In contrast, SalR expression was low in spe- CYP80, CYP82, and CYP94 families. Many of these P450-dependent cies that do not accumulate morphinan or promorphinan alkaloids. monooxygenases might catalyze unique reactions responsible for However, cDNAs coding for proteins with more than 95% amino the unique alkaloid profiles of different Papaver species. These spe- acid identity to P. somniferum SalR could be isolated from several cies have been reported to accumulate aporphine, protoberberine, Papaver species. Furthermore, the heterologously expressed SalR and rhoeadine alkaloids, which are extensively ring hydroxylated homologues from P. arenarium, P. bracteatum, P. pilosum and P. and frequently contain methylenedioxy bridges. pyrenaicum showed salutaridine reductase activity (Ziegler et al., unpublished). 3. Characterization of cDNAs involved in BIA metabolism based The occurrence of active SalR homologues in Papaver pilosum on transcriptional profiles and P. pyrenaicum was unexpected considering that both species do not produce salutaridine or any other promorphinan alkaloid. The integration of alkaloid and transcript profiles was aimed at Since BIA metabolism is active in all species, the factor determining identifying cDNAs potentially involved in the formation of specific the capacity for promorphinan alkaloid production seems to occur BIAs. The number of candidate cDNAs was reduced to about 100 by between (S)-reticuline and salutaridine. It will be interesting to transcript profile analysis. As most of the cDNAs encoding BIA bio- determine whether or not all species devoid of promorphinan alka- synthetic enzymes were clustered, we first focused on uncharac- loids can catalyze the inversion of stereochemistry from (S)-reticu- terized cDNAs present in the cluster. The full-length cDNA for line to (R)-reticuline, or whether they contain an active EST A21G11 was highly similar to 6OMT. Subsequent characteriza- salutaridine synthase. The formation of a multienzyme complex tion of the recombinant protein identified this cDNA as a novel and involving several promorphinan- and morphinan-specific enzymes highly specific norreticuline 7-O-metyltransferase (N7OMT; Pie- can also not be ruled out. The alkaloid profiles of transgenic P. som- nkny et al., 2009). The higher abundance of N7OMT transcripts in niferum plants in which the levels of morphine biosynthetic en- some plants correlated with the accumulation of papaverine and zymes have been suppressed suggested the potential suggested its involvement in papaverine biosynthesis. The EST involvement of metabolic channels. Silencing of SalAT led to the CAG-3 showed high homology to P450-dependent monooxygena- accumulation of salutaridine rather than of the substrate salutarid- ses of the CYP719 family. Biochemical characterization of the re- inol (Allen et al., 2008; Kempe et al., 2009), and suppression of combinant enzyme identified this P450 enzyme as salutaridine CoR1 expression resulted in accumulation of reticuline (Allen synthase (Gesell et al., 2009). et al., 2004). However, it was not determined which reticuline The EST 16B1 showed high homology to members of the short enantiomer was produced. chain dehydrogenase/reductase (SDR) family. The amino acid sequence was most similar to a SDR from Arabidopsis thaliana, fol- 5. Structure-function analysis of SalR lowed by SDRs involved in menthol biosynthesis. This cDNA could encode an enzyme involved in one of three reductive steps SalR is a member of the SDR family of enzymes, which cata- in morphine biosynthesis, including the reduction of (1) 1,2- lyze NAD(P)(H)-dependent oxidation/reduction reactions and dehydroreticulinium ion to (R)-reticuline, (2) salutaridine to consist of a one-domain subunit of about 250 amino acids with 7(S)-salutaridinol, and (3) codeinone to codeine. Ultimately, the the cofactor and substrate binding sites in the N-terminal and C- recombinant protein from P. somniferum was shown to catalyze terminal domains, respectively (Jörnvall et al., 1995). A main fea- the stereospecific reduction of salutaridine to 7(S)-salutaridinol ture of SDR proteins are the highly conserved TGxxxGhG (h indi- (Ziegler et al., 2006). No activity could be detected with 1,2-dehy- cates any hydrophobic residue) motif for coenzyme binding and droreticulinium ion or codeine, or with other potential substrates the YxxxK motif, which together with an upstream serine resi- including tropinone or ()-menthone. The enzyme displayed a due comprises the catalytic center (Oppermann et al., 1997). strong preference for NADPH compared with NADH, and was able Additionally, an asparagine residue has been proposed as part to catalyze the reverse reaction from salutaridinol to salutaridine of a catalytic tetrad through positioning the lysine side chain, at higher pH. The same properties have been reported for salu- thereby forming a proton relay system (Filling et al., 2002). Most taridine reductase from P. somniferum cell culture extracts (Gerar- of the SDRs involved in plant secondary metabolism are multi- dy and Zenk, 1993b). The similarity with the partially purified meric proteins consisting of two or four 28 kDa subunits. SalR protein from plant extracts as well as the higher expression levels belongs to a new class of SDRs since it is active as a monomer of of the gene corresponding to EST 16B1 in morphinan and promo- 35 kDa. The increased length is attributable to an insertion of rphinan alkaloid-accumulating plants supported the identification 40 amino acids upstream of the catalytic YxxxK motif. This fea- of the enzyme as salutaridine reductase. The stereospecificity of ture is shared by other plant SDRs which catalyze reductions in this enzyme is of major importance, since only 7(S)-salutaridinol monoterpenoid metabolism (Davis et al., 2005; Ringer et al., is acetylated by the next step in morphine biosynthesis, salutari- 2003). These proteins are closely related to monomeric SDRs 1704 J. Ziegler et al. / Phytochemistry 70 (2009) 1696–1707

Fig. 5. Comparison of P. bracteatum SalR-2 to monomeric SDRs (A) Amino acid sequence alignments of selected monomeric SDRs. The sequence alignment was performed using the CLUSTALW application (Thompson et al., 1994) of MegAlign (DNASTAR Inc.). The protein sequence of P. bracteatum SalR (Pb-SalR; GenBank accession number EF184229) was aligned to M. x piperita ()-menthone:()-menthol reductase (Mp-MMR; AAQ55960), M. x piperita ()-menthone:(+)-neomenthol reductase (MpMNR1; AAQ55959), M. x piperita ()-menthone:(+)-neomenthol reductase (Mp-MNR2; DQ362936), A. thaliana At3g61220 gene product (At3g61220; NM115986), porcine testicular carbonyl reductase (PTCR; NP999238), C. annuum MNR1 (Ca-MNR; EF576664), and to the monomeric SDRs of BIA-producing cell cultures from Nandina domestica (Nd-12F11; FJ789568), P. bracteatum (Pb-2F1; FJ789569), and C. majus (Cm-39E4; FJ789567). The areas containing amino acid residues involved in salutaridine binding in Pb-SalR as well as the cofactor binding motif TGxxxGhG are boxed. Solid triangles denote the catalytic residues for SDRs. (B) Neighbour-joining phylogenetic tree of functionally characterized SDRs involved in plant secondary metabolism. The amino acid alignment was performed as described in (A). The tree was generated and visualized with the TREECON software (Yves van de Peer, University of Konstanz, Germany). Bootstrap values in per cent of 1000 trials are indicated. Hn-TRI: Hyoscyamus niger tropinone reductase I (BAA85844). from mammals, such as the multifunctional porcine testicular anism of substrate binding, homology modelling of SalR was carbonyl reductase (PTCR) or human 15-hydroprostaglandine performed and salutaridine was docked into the active site. In dehydrogenase/carbonyl reductase (Ghosh et al., 2001; Tanaka order to facilitate site-directed mutagenesis experiments fol- et al., 2005). The crystal structure of the mammalian proteins lowed by biochemical characterization of mutated proteins, SalR showed that the additional amino acids form an a-helix that from P. bracteatum instead from P. somniferum was modelled, blocks the dimerization interface, leading to their monomeric since the protein from P. bracteatum could be puriﬁed from bac- nature (Ghosh et al., 2001). However, the monomeric plant SDRs teria in much higher amounts. The model of P. bracteatum SalR show an additional 40 amino acid insertion compared to the consists of six parallel b-sheets, each of which is ﬂanked on both mammalian enzymes (Fig. 5A). In order to investigate the signif- sides by three parallel a-helices (Fig. 6A). The aA–aF segment icance of the additional amino acids and to elucidate the mech- represents a double tortuous a/b-motif with alternating b-sheets J. Ziegler et al. / Phytochemistry 70 (2009) 1696–1707 1705

Fig. 6. Homology model and substrate binding site P. bracteatum SalR (A) Ribbon diagram of the P. bracteatum SalR model. Turquoise: a-helices; magenta: b-sheets; blue: aE and aF helices involved in dimerization in dimeric SDR proteins; orange: aF0-1 to aF0-4 helices suggested to prevent dimerization; red: aE0 helix. Salutaridine and NADPH are depicted in green and orange, respectively. (B) Salutaridine binding of P. bracteatum SalR. The aE0 helix is shown in red. The colours for the carbon skeleton of each structure are green for salutaridine, and orange for amino acids. Nitrogen, oxygen, and sulphur atoms are shown in blue, red, and yellow, respectively. Hydrogen bonds are depicted as broken lines. Hydrogen atoms have been omitted for clarity. The areas indicated in Fig. 5, consist of F104, V106, D107 (area I); S181, T182, L185, K186 (area II); L266, M271, N272, I275 (area III). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) and a-helices and forms the classical Rossman fold for cofactor 50% of the residues in the substrate binding site are identical binding typical for SDRs. Similar to the mammalian proteins, (Fig. 5A). Interestingly, the putative binding sites of enzymes that the additional 40 amino acids specific to monomeric SDRs form convert ()-menthone are variable. Mp-MMR contains several helices that obstruct access to the surface for the interaction of amino acid substitutions compared with Mp-MNR1 or Mp-MNR2, dimeric SDRs (Ghosh et al., 2001). One segment of 14 amino which might account for the differential product specificity. acids, which are part of the additional amino acids in plant However, also Mp-MNR1 and Mp-MNR2 show only low sequence monomeric SDRs, forms an a-helix, named aE’ (Geissler et al., identity in these regions despite catalyzing the same reaction with 2007). Substrate docking and site-directed mutagenesis revealed ()-menthone (Fig. 5A). that the amino acids forming the substrate binding site reside in Considering the occurrence of identical amino acid residues, the three areas (Geissler et al., 2007). Substrate affinity is mainly putative substrate binding sites in Nd-12F11, Cm-39E4, determined by amino acids in area I adjacent to the new a helix At3g61220, and Ca-MNR, all of which convert ()-menthone to (Phe-104, Val-106, and Asp-107), and area III (Leu-266, Met-271, (+)-neomenthol, are more similar to SalR than to enzymes from Asn-272, and Ile-275). The stabilization of the correct position of M. x piperita. Although the substrate binding sites are not necessar- the substrate is achieved mainly by residues from area II (Ser- ily in the same relative location compared with SalR, three sub- 181, Thr-182, Leu-185, and Lys-186), which form the bottom of stantially different ()-menthone-binding sites might occur in the substrate binding sites (Fig. 6B). (+)-neomenthol-producing enzymes. Preliminary site-directed mutagenesis analysis suggests that the ()-menthone-binding re- 6. Relationship of SalR to other SDRs gion in Mp-MNR2 and the salutaridine-binding domain in SalR are in the same relative position. SalR exhibits similarities of 42% and 47% on amino acid level to The occurrence of different ()-menthone-binding sites might ()-menthone:()-menthol reductase (Mp-MMR) and two ()- be explained by the source of the enzymes and their putative role menthone:(+)-neomenthol reductases (Mp-MNR1 and Mp- in vivo. M. x piperita produces ()-menthone, ()-menthol and (+)- MNR2), respectively, from Mentha x piperita (Fig. 5)(Davis et al., neomenthol, whereas these products have not been detected in 2005; Ziegler et al., 2006). Higher similarities are observed to Arabidopsis, N. domestica, C. majus,orC. annuum (Choi et al., monomeric SDRs from A. thaliana (At3g61220; 54%) and Capsicum 2008). Thus, it is likely that ()-menthone reduction represents a annuum (Ca-MNR; 50%), both of which exhibit ()-menthone:(+)- common, non-specific side activity of these enzymes, and that they neomenthol reductase (MNR) activity (Ziegler et al., 2006; Choi did not evolve to perform this task. However, the actual in vivo et al., 2008). In order to compare SDR homologues from more clo- substrates are not known. sely related species, we searched the cell culture EST databases of Interestingly, the ()-menthone reductase genes from Arabid- several BIA-producing species. Three cDNAs from different species opsis and C. annuum have recently been shown to participate in were obtained: Nandina domestica 12F11 (Nd-12F11; 59% the protection of these plants against pathogens (Choi et al., homology to Pb-SalR), Chelidonium majus (Cm-39E4; 59%), and 2008). Silencing of Ca-MNR in C. annuum rendered the plant more P. bracteatum (Pb-2F1; 72%). Nd-12F11 as well as Cm-39E4 protein susceptible to Xanthomonas campestris pv. vesicatoria and Colleto- showed MNR activity, but were unable to reduce salutaridine. Pb- trichum coccodes. Overexpression of the gene in Arabidopsis led 2F1 exhibited no activity with ()-menthone or salutaridine as to enhanced resistance towards Pseudomonas syringae pv. tomato substrates (Ziegler and Facchini, unpublished). Furthermore, DC3000 and Hyaloperonospera parasitica isolate Noco2, whereas At3g61220 and Mp-MNR2 do not accept salutaridine (Ziegler Arabidopsis T-DNA insertion lines affecting At3g61220 expression et al., 2006). were more susceptible. In both cases, enhanced resistance was Considering the amino acids responsible for salutaridine bind- accompanied by increased expression of pathogenesis-related ing in P. bracteatum SalR, substantial changes were apparent in genes. Although the in planta role of the enzymes is not known, the other SDRs explaining why they are unable to reduce salutari- they were suggested to confer pathogen resistance by detoxifica- dine, and why SalR does not accept ()-menthone. Even for Pb-2F1, tion of xenobiotic compounds produced by pathogens. Alterna- which shares 72% overall amino acid identity with SalR, less than tively, they could convert inactive endogenous compounds into 1706 J. Ziegler et al. / Phytochemistry 70 (2009) 1696–1707 active pathogen inhibitors, or activate defence genes (Choi et al., Geissler, R., Brandt, W., Ziegler, J., 2007. Molecular modeling and site-directed 2008). mutagenesis reveal the benzylisoquinoline binding site of the short-chain dehydrogenase/reductase salutaridine reductase. Plant Physiol. 143, 1493– Ca-MNR and At3g61220 showed similar sequence identities 1503. (between 50% and 55%) to ()-menthone reductases from M. x Gerardy, R., Zenk, M.H., 1993a. Formation of salutaridine from (R)-reticuline by a piperita, and to SalR from Papaver species (Fig. 5). We suggest that membrane bound cytochrome P450 enzyme from Papaver somniferum. Phytochemistry 32, 79–86. ()-menthone reductases from M. x piperita and SalR from Papaver Gerardy, R., Zenk, M.H., 1993b. Purification and characterization of salutaridine: species might have a common ancestor potentially involved in NADPH 7-oxidoreductase from Papaver somniferum. Phytochemistry 34, 125– pathogen defence. Speciation of an ancestral gene that unspecifi- 132. Gesell, A., Rolf, M., Ziegler, J., Diaz-Chavez, M.L., Huang, F.C., Kutchan, T.M., 2009. cally reduced ()-menthone to (+)-neomenthol would have led CYP719B1 is salutaridine synthase, the C-C phenol-coupling enzyme of to specific ()-menthol producing reductases and salutaridine morphine biosynthesis in opium poppy. J. Biol. Chem. 2009, doi:10.1074/ reductase. A similar scenario has been observed for norcoclaurine jbc.M109.033373. Ghosh, D., Sawicki, M., Pletnev, V., Erman, M., Ohno, S., Nakajin, S., Duax, W.L., 2001. synthase, the first enzyme of BIA biosynthesis. One of the two en- Porcine carbonyl reductase structural basis for a functional monomer in short zymes, which can perform this reaction, is closely related to the chain dehydrogenases/reductases. J. Biol. Chem. 276, 18457–18463. PR10 family of pathogenesis-related proteins and to the Betv1 Grothe, T., Lenz, R., Kutchan, T.M., 2001. 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