Evolution of rosmarinic acid biosynthesis · Review Evolution of rosmarinic acid biosynthesis Maike...

Phytochemistry 70 (2009) 1663–1679

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Phytochemistry

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Review

Evolution of rosmarinic acid biosynthesis

Maike Petersen *, Yana Abdullah, Johannes Benner, David Eberle, Katja Gehlen, Stephanie Hücherig,Verena Janiak, Kyung Hee Kim, Marion Sander, Corinna Weitzel, Stefan WoltersInstitut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstr. 17A, D-35037 Marburg, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 February 2009Received in revised form 19 May 2009Available online 25 June 2009

Keywords:Rosmarinic acidCaffeoylshikimic acidChlorogenic acidPhenylpropanoid metabolismAcyltransferaseCYP98A

0031-9422/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.phytochem.2009.05.010

* Corresponding author. Tel.: +49 6421 2825821; faE-mail address: [email protected] (M

Rosmarinic acid and chlorogenic acid are caffeic acid esters widely found in the plant kingdom and pre-sumably accumulated as defense compounds. In a survey, more than 240 plant species have beenscreened for the presence of rosmarinic and chlorogenic acids. Several rosmarinic acid-containing specieshave been detected. The rosmarinic acid accumulation in species of the Marantaceae has not been knownbefore. Rosmarinic acid is found in hornworts, in the fern family Blechnaceae and in species of severalorders of mono- and dicotyledonous angiosperms. The biosyntheses of caffeoylshikimate, chlorogenicacid and rosmarinic acid use 4-coumaroyl-CoA from the general phenylpropanoid pathway as hydroxy-cinnamoyl donor. The hydroxycinnamoyl acceptor substrate comes from the shikimate pathway: shiki-mic acid, quinic acid and hydroxyphenyllactic acid derived from L-tyrosine. Similar steps are involvedin the biosyntheses of rosmarinic, chlorogenic and caffeoylshikimic acids: the transfer of the 4-coumaroylmoiety to an acceptor molecule by a hydroxycinnamoyltransferase from the BAHD acyltransferase familyand the meta-hydroxylation of the 4-coumaroyl moiety in the ester by a cytochrome P450 monooxygen-ase from the CYP98A family. The hydroxycinnamoyltransferases as well as the meta-hydroxylases showhigh sequence similarities and thus seem to be closely related. The hydroxycinnamoyltransferase andCYP98A14 from Coleus blumei (Lamiaceae) are nevertheless specific for substrates involved in RA biosyn-thesis showing an evolutionary diversification in phenolic ester metabolism. Our current view is that onlya few enzymes had to be ‘‘invented” for rosmarinic acid biosynthesis probably on the basis of genesneeded for the formation of chlorogenic and caffeoylshikimic acid while further biosynthetic steps mighthave been recruited from phenylpropanoid metabolism, tocopherol/plastoquinone biosynthesis andphotorespiration.

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Contents

1. Rosmarinic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1664

1.1. Occurrence of rosmarinic acid in the plant kingdom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16641.2. Screening of plant species for the presence of rosmarinic acid and chlorogenic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1665

2. Biosynthesis of rosmarinic acid, chlorogenic acid and caffeoylshikimic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16663. D-isomer-specific 2-hydroxyacid dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1672

3.1. Hydroxy(phenyl)pyruvate reductase from C. blumei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1673

4. CoA-ester-dependent BAHD hydroxycinnamoyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1673

4.1. Hydroxycinnamoyltransferases in phenolic metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16734.2. Properties of hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase (rosmarinic acid synthase; RAS) . . . . . . . . 1675

5. Cytochrome P450 CYP98A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1675

5.1. CYP98A in phenylpropanoid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16755.2. Properties of 4-coumaroyl-4’-hydroxyphenyllactate 3- and 3’-hydroxylase from C. blumei (CYP98A14) . . . . . . . . . . . . . . . . . . . . . . . . . 1676

6. Possible evolutionary relationship of caffeoylshikimic/chlorogenic acid and rosmarinic acid biosynthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1676Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1677References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1677

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x: +49 6421 2825828.. Petersen).

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COOH

NH2

COOH

NH2HO

COOH

COOH

HOO

COOH

HO

COOH

HOHO H

HO

O

SCoA

PAL

C4H

4CL

TAT

HPPR

O

HCOOHO

HO

OH

O

HCOOHO

HO

OH

O

HCOOHO

HO

OH

HOOH

O

HCOOHO

HO

OH

HOOH

4C-DHPL 3H

RAS

4-coumaroyl-CoA

4-coumaric acid

4-hydroxyphenylpyruvic acid

4-hydroxyphenyllactic acid

L-phenylalanine L-tyrosine

t-cinnamic acid

4-coumaroyl-4'-hydroxyphenyllactic acid

caffeoyl-4'-hydroxy- 4-coumaroyl-3',4'-dihydroxy-

rosmarinic acid

HPPDhomogentisic acid

tocopherols

plastoquinones

HO4-coumaroyl-5'-O-quinic acid

O

OCOOH

OH

HO

OH

HO

caffeoyl-5'-O-quinic acid = chlorogenic acid

O

OCOOH

OH

HO

OH

HO

4C-S/Q 3H

+ quinic acid

+ shikimic acid

HO4-coumaroyl-5'-O-shikimic acid

O

O

HO

OH

COOH

HOcaffeoyl-5'-O-shikimic acid

O

O

HO

OH

COOHHO

caffeoyl-CoA + shikimic acidHCS/QT (?)

HCS/QT

4C-S/Q 3H

4C-pHPL 3'H

Caf-pHPL 3'H

4C-pHPL 3H

phenyllactic acid phenyllactic acid

+ CoA

Fig. 1. Proposed biosynthetic pathways to rosmarinic acid, caffeoylshikimic acid and chlorogenic acid as well as some other phenylpropanoid pathway-derived compounds.The involved enzymes are: PAL = phenylalanine ammonia lyase; C4H = cinnamic acid 4-hydroxylase; 4CL = 4-coumaric acid CoA-ligase; TAT = tyrosine aminotransferase;HPPR = hydroxyphenylpyruvate reductase; RAS = ‘‘rosmarinic acid synthase”, hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase; 4C-pHPL 3H, 4C-pHPL 30H = 4-coumaroyl-40-hydroxyphenyllactate 3/30-hydroxylases; Caf-pHPL 30H = caffeoyl-40-hydroxyphenyllactate 30-hydroxylase; 4C-DHPL 3H = 4-coumaroyl-30 ,40-dihydroxyphenyllactate 3-hydroxylase; HPPD = hydroxyphenylpyruvate dioxygenase; HCS/QT = hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase;4C-S/Q 3H = 4-coumaroylshikimate/quinate 3-hydroxylase.

1664 M. Petersen et al. / Phytochemistry 70 (2009) 1663–1679

1. Rosmarinic acid

1.1. Occurrence of rosmarinic acid in the plant kingdom

The tanning compounds of Labiates have been known as Labiatetannins (‘‘Labiatengerbstoffe”) for a long time. Research into theidentification of these compounds started around 1950, and a com-pound named rosmarinic acid (RA) was isolated from Rosmarinusofficinalis (Lamiaceae) by Scarpati and Oriente (1958). Rosmarinicacid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid(Fig. 1). It was isolated from many species of the families Lamia-ceae and Boraginaceae and was identified as one of the active com-ponents of several medicinal plants (e.g. Salvia officinalis, Mentha xpiperita, Thymus vulgaris, Melissa officinalis, Symphytum officinale)within these families. Not all members of the Lamiaceae, however,contain RA. The occurrence is mainly restricted to the subfamilyNepetoideae (Litvinenko et al., 1975), which is regarded as mono-

phyletic according to Grayer et al. (2003). The presence of RA in theLamiaceae outside of the subfamily Nepetoideae was, however, re-ported by Pedersen (2000) in Teucrium scorodonia and Aegiphilamollis of the sub-family Teucrioideae and Hymenopyramis brachiataof uncertain affinity within the family (according to Olmstead(2005)). On the other hand, RA was found in many species outsidethe Lamiaceae and Boraginaceae (Petersen and Simmonds, 2003;Table 1). The use of RA occurrence as a chemotaxonomical markertherefore is not recommendable. As can be seen from Table 1, RA isalready present in the Anthocerotaceae (hornworts), one of theearliest land plant families. Own unpublished investigations couldnot show the occurrence of RA in the genus Chara of the green al-gae family Charophyceae which is seen as the algal predecessor ofland plants. Within the land plants RA was found in species of thehornworts (family Anthocerotaceae), ferns (family Blechnaceae) aswell as some orders of the monocotyledonous plants and therosids and asterids within the eudicotyledonous plants (Fig. 2).

Table 1Published occurrence of rosmarinic acid in the plant kingdom. Systematics are according to ‘‘Strasburger – Lehrbuch der Botanik” (Bresinsky et al., 2008).

Subclass Order Family Reference

AnthocerophytinaAnthocerotales Anthocerotaceae Takeda et al. (1990), Petersen (2003) and Vogelsang et al. (2005)

FilicophytinaBlechnales Blechnaceae Harborne (1966), Bohm (1968) and Häusler et al. (1992)

Spermatophytina, class MagnoliopsidaBasal orders Chloranthales Chloranthaceae Zhu et al. (2008)Monocoty ledonous

plantsAlismatales Araceae Aquino et al. (2001)

Potamogetonaceae Petersen (unpublished)Zosteraceae Ravn et al. (1994) and Achamlale et al. (2009)

Zingiberales Cannaceae Petersen and Simmonds (2003) and Yun et al. (2004)Marantaceae Abdullah et al. (2008)

Liliales Melianthaceae Lee et al. (2008)Eudicoty ledons Myrtales Onagraceae Huang et al. (2007)

Celastrales Celastraceae Ly et al. (2006)Rosales Rosaceae Amzad Hossain et al. (2009)Cucurbitales Cucurbitaceae De Tommasi et al. (1991)Malvales Malvaceae/Sterculiaceae Satake et al. (1999)

Malvaceae/Tiliaceae Lasure et al. (1994) and Ho et al. (1995)Gentianales Rubiaceae Aquino et al. (1990)Lamiales Lamiaceae Scarpati and Oriente (1958), Litvinenko et al. (1975), Janicsak et al. (1999) and Pedersen (2000)

Plantaginaceae Kurkin et al. (1988) cited in Holzmannova (1995) and Velazquez Fiz et al. (2000)Acanthaceae Harborne (1966)Scrophulariaceae Fernandez et al. (1995)

Unclear Boraginaceae/Hydrophyllaceae

Kelley et al. (1975), Harborne (1966) and Petersen (unpublished)

Apiales Apiaceae Hiller (1965), Hiller and Kothe (1967), Parejo et al. (2004), Le Claire et al. (2005), Yoshida et al. (2005)and Olivier et al. (2008)

Araliaceae Trute and Nahrstedt (1996)Asterales Asteraceae Rubio et al. (1992)Dipsacales Dipsacaceae Kowalczyk (1996)

M. Petersen et al. / Phytochemistry 70 (2009) 1663–1679 1665

Interestingly there is no report about RA in gymnosperms and onlyone recent finding of RA derivatives in the basal dicot order Chlo-ranthales in Sarcandra glabra (Chloranthaceae; Zhu et al., 2008).

1.2. Screening of plant species for the presence of rosmarinic acid andchlorogenic acid

Although several publications have dealt with the detection ofRA in members of various plant families (see e.g. Litvinenkoet al., 1975; Holzmannová, 1995; Janicsák et al., 1999; Pedersen,2000, and others) many other reports only deal with one or afew species. We have therefore set out to screen plant species col-lected from the wild and from botanical gardens for their RA andchlorogenic acid (CA) content. Accumulation of RA or CA is notmutually exclusive. Many species from several families containboth phenolic compounds (Table 2).

The earliest land plants with RA occurrence are the hornwortgenera Anthoceros and Folioceros (Anthocerotaceae, Anthocero-tales) which are phylogenetically closely related to each other(Takeda et al., 1990; Petersen, 2003; Vogelsang et al., 2005 andown unpublished results). Within the pteridophytes, species ofthe genus Blechnum (Blechnaceae, Blechnales) (Harborne, 1966;Bohm, 1968; Häusler et al., 1992 and own unpublished results)definitely accumulate RA. Interestingly, of seven examined Blech-num species only two showed RA accumulation. In a survey byBohm (1968) only Blechnum brasiliense contained RA, while allother examined 39 eusporangiate and leptosporangiate fern spe-cies were devoid of this phenolic compound, nine of the investi-gated fern species contained CA.

The presence of RA in Sarcandra glabra (Chloranthaceae; Zhuet al., 2008) belonging to the primitive angiosperm order Chlorant-hales prompted us to analyse two Chloranthus species. Leaves ofChloranthus officinalis showed the presence of RA and CA, whileChloranthus spicatus only contained CA.

RA has been detected in three orders of the monocotyledonousplants, namely Alismatales, Liliales and Zingiberales (Ravn et al.,1994; Aquino et al., 2001; Petersen and Simmonds, 2003; Yunet al., 2004; Lee et al., 2008, and own unpublished results). Re-cently, the occurrence of RA in Marantaceae species was shownfor the first time. Here again the occurrence of RA was not a consis-tent feature of all species of this family (Abdullah et al., 2008).Within the order Zingiberales, RA was also detected in the Canna-ceae family (Petersen and Simmonds, 2003; Yun et al., 2004) whichis a near sister family to the Marantaceae (Kress et al., 2001).

Within the eudicotyledonous plants RA has been found in manyorders of the Rosids and the Asterids and thus in the more evolvedorders (Fig. 2, Table 2). In general, however, our own screeningonly showed RA accumulation in families that have already beendescribed previously to have species with RA accumulation. Addi-tional species of these groups have been detected in our screening.RA as natural product is well-known from Lamiaceae species. How-ever, a massive presence of RA-accumulating species is only ob-served in the subfamily Nepetoideae while members of theTeucrioideae and Scutellarioideae (as shown in Table 2) and theother subfamilies mostly are devoid of RA. RA accumulation seemsto be a rather consistent feature of species within the Boraginaceaeincluding the formerly separate family Hydrophyllaceae (Table 2).Several species of the Apiaceae, namely Centella asiatica, Saniculaeuropaea, Eryngium alpinum and Foeniculum vulgare, accumulateRA (Hiller, 1965; Hiller and Kothe, 1967; Parejo et al., 2004; LeClaire et al., 2005; Yoshida et al., 2005). We now found Astrantiamajor as a new RA-forming species of this family. Helicteres isora(Malvaceae, formerly: Sterculiaceae) has been reported to accumu-late RA as well as the less hydroxylated precursor isorinic acid(Satake et al., 1999). Helicteres jamaicensis, but no other examinedspecies of the Malvaceae, contained RA in our study.

All previous studies as well as our own investigations show arather scattered occurrence of RA. Moreover, RA is present in plant

Fig. 2. Phylogenetic relationship in angiosperms (redrawn from Bresinksky et al. (2008)). Orders with members reported to contain RA are marked in bold print.


species of one of the earliest groups of land plants (hornworts) upto highly evolved species of the monocotyledonous and eudicoty-ledonous plants. Our investigations might – in the long run – givesome explanation for this observation.

Chlorogenic acid (CA; caffeoyl-50-O-quinic acid) is more widelydistributed in the plant kingdom. It was first detected in 1837 incoffee (Sondheimer, 1964) and is an ester of caffeic acid and (L)-quinic acid (Fig. 1) that can occur alone or along with RA (Table2). Several isomeric caffeoylquinic acid esters (30-/40-O-caffeoylqui-nic acid) as well as several dicaffeoylquinic acids (e.g. 10,30-dica-ffeoylquinic acid = cynarin from Cynara scolymus) are knownfrom the plant kingdom. In our investigation, CA was found in sev-eral Blechnaceae species and was abundant in species of the ordersApiales, Asterales and Dipsacales, whereas the family Boraginaceaeonly shows a scattered occurrence of CA. CA was only rarely de-tected in monocotyledonous plants and in most cases concomi-tantly with RA.

2. Biosynthesis of rosmarinic acid, chlorogenic acid andcaffeoylshikimic acid

Enzymes involved in the biosynthesis of RA have been eluci-dated in several plant species of the Lamiaceae and Boraginaceae,namely Coleus blumei (Lamiaceae; Petersen et al., 1993; Petersen,1997), M. officinalis (Lamiaceae; Weitzel and Petersen, unpub-lished), Anchusa officinalis (Boraginaceae; De-Eknamkul and Ellis,1987), Salvia miltiorrhiza (Lamiaceae; Yan et al., 2006; Huanget al., 2008) and Lithospermum erythrorhizon (Boraginaceae;Yamamura et al., 2001; Matsuno et al., 2002). The cDNAs encodingseveral biosynthetic enzymes have been isolated as well.

The biosynthesis as shown in C. blumei (Fig. 1; Petersen et al.,1993; Petersen, 1997; Petersen and Simmonds, 2003 and literaturecited therein) starts with the aromatic amino acids L-phenylalanineand L-tyrosine which are separately transformed to the intermedi-ary precursors 4-coumaroyl-CoA and 4-hydroxyphenyllactic acid.

Table 2Screening of the plant kingdom for the occurrence of rosmarinic acid (RA) and chlorogenic acid (CA). The presence of the compounds in 70% ethanolic extracts from leaves was verified byHPLC and TLC as described by Abdullah et al. (2008). The plant material was collected in different botanical gardens: D = Botanical Garden of the Heinrich-Heine-Universität Düsseldorf,G = Botanical Garden of the Justus-Liebig-Universität Giessen, K = Botanical Garden of the Christian-Albrechts-Universität Kiel, M = Botanical Garden of the Philipps-UniversitätMarburg, T = Institut für Pharmazeutische Biologie der Eberhard-Karls-Universität Tübingen. In the family Lamiaceae, subfamilies (as defined by Olmstead, 2005) are indicated:NE = Nepetoideae, TE = Teucrioideae, SC = Scutellarioideae. For species marked with an asterisk (*) the occurrence of RA has – to our best knowledge – not been reported before.

Family Genus or species Source RA CA

Ferns

Equisetaceae Equisetum arvense D – n.d.

Order: BlechnalesBlechnaceae Blechnum arcuatum M – +

Blechnum brasiliense M + +Blechnum gibbum* M + +Blechnum occidentale M – +Blechnum penna-marina D – n.d.Blechnum polypodioides M – –Blechnum spicant M – n.d.Doodia caudata G – –

‘‘Basal orders”Order: Chloranthales

Chloranthaceae Chloranthus officinalis * M + +Chloranthus spicatus M – +

Monocotyledonous plantsOrder: Alismatales

Araceae Arum italicum M – –Juncaginaceae Triglochin striatum M – –Zosteraceae Zostera marina - + n.d.Potamogetonaceae Potamogeton coloratus* D + n.d.

Order: LilialesHyacinthaceae Chionodoxa luciliae M – –

Order: PoalesJuncaceae Juncus effusus M – –

Juncus subnodulosus M – +

Order: ZingiberalesCannaceae Canna edulis M + +

Canna indica M + +Costaceae Costus cuspidatus M – –Heliconiaceae Heliconia humilis M – –

Heliconia jacquinii M – –Lowiaceae Orchidantha maxillarioides M – –Musaceae Musa acuminata M – –

Musa textilis M – –Marantaceae Ataenidia conferta K – –

Calathea argyraea G – –Calathea bachemiana G – –Calathea insignis M – –Calathea kegeljani M – –Calathea lancifolia G – –Calathea lindeniana M – –Calathea lietzei G – –Calathea louisae M – –Calathea makoyana M – –Calathea mediopicta G – –Calathea micans M – –Calathea ornata M – –Calathea picturata G – –Calathea rotundifolia K – –Calathea undulata G – –Calathea variegata M – –Calathea warscewiczii K – –Calathea zebrina M – –Ctenanthe burle-marxii M – –Ctenanthe kummeriana G – –Ctenanthe lubbersiana G – –Ctenanthe pilosa K – –Ctenanthe setosa K – –Maranta arundinacea K – –Maranta depressa* K + +Maranta leuconeura ‘‘Fascinator”* M + +Maranta leuconeura var. kerchoviana* M + +Maranta leuconeura var. massangeana* G + +Maranta noctiflora G – –Marantochloa flexuosa M – –

(continued on next page)


Table 2 (continued)


Megaphrynium macrostachyum M – –Pleiostachya pruinosa M – –Stromanthe amabilis G – +Stromanthe sanguinea G – –Thalia dealbata M – –Thalia geniculata* M + –

Strelitziaceae Strelitzia reginae M – –Zingiberaceae Curcuma longa M – –

Globba marantina M – –Zingiber officinale M – –

Eudicotyledonous plantsOrder: Geraniales

Geraniaceae Erodium manescavii M – –Geranium sanguineum M – –Geranium swatense M – –Geranium sylvaticum M – +

Order: MyrtalesLythraceae Cuphea procumbens M – –

Lythrum alatum M – –Lythrum hyssopifolia M – –

Onagraceae Gaura biennis M – –Oenothera missouriensis M – –Lopezia racemosa M – –

Order: CelastralesAquifoliaceae Ilex aquifolium M – +Celastraceae Euonymus verrucosus M – –

Order: RosalesHydrangeaceae Philadelphus spec. M – +

Order: CucurbitalesCucurbitaceae Cucurbita pepo var. giromontiina M – –

Cucurbita pepo var. oleifera M – –

Order: BrassicalesBrassicaceae Arabidopsis thaliana M – n.d.

Order: MalvalesMalvaceae incl. Sterculiaceae, Tiliaceae Abroma augustum M – –

Abutilon theophrasti M – –Alcea rosea M – –Alyogyne huegelii M – –Callirhoe involucrata M – –Dombeya rotundifolia M – –Helicteres jamaicensis* M + –Hibiscus cannabinus M – –Hibiscus rosa-sinensis M – –Lavatera trimestris M – –Malope trifida M – –Pavonia X gledhillii M – –Theobroma cacao M – +Tilia americana M – –Tilia amurensis M – –Tilia cordata M – +Tilia japonica M – +Tilia platyphyllos M – +Tilia tomentosa M – –

Thymelaeaceae Daphne mezereum M – –

Order: CornalesCornaceae Cornus alba M – –

Cornus mas M – –Cornus officinalis M – –

Order: GentianalesApocynaceae Catharanthus roseus M – +Asclepiadaceae Asclepias syriaca M – –Gentianaceae Gentiana asclepiadea M – –

Gentiana lutea M – –Rubiaceae Galium boreale M – +

Galium rubioides M – +Phuopsis stylosa M – –Rubia tinctoria M – +

Order: LamialesAcanthaceae Acanthus hungaricus M – –

Acanthus longifolius M – –Barleria micans M – +


Table 2 (continued)


Jacobinia zelandia M – –Odontonema schomburgkianum M – –

Gesneriaceae Phinaea multiflora M – –Streptocarpus caulescens M – –Streptocarpus rexii M – –

Lamiaceae Ajuga chamaepitys TE D – n.d.Agastache rugosa NE D + n.d.Calamintha nepeta NE M + –Cedronella canariensis NE D + n.d.Coleus forskohlii (syn. Plectranthus barbatus) NE T + n.d.Collinsonia canadensis NE M + –Dracocephalum spec. NE M + +Elsholtzia stauntonii* NE D + n.d.Glechoma hederacea NE M + +Hormium pyrenaicum* NE D + n.d.Lavandula angustifolia NE M + –Lavandula multifida NE M + –Lycopus europaeus NE - + n.d.Lycopus exaltatus NE D + n.d.Melissa officinalis NE M + –Mentha aquatica NE M + –Micromeria thymifolia NE M + –Monarda punctata* NE D + n.d.Origanum majorana NE M + –Perilla frutescens NE D + n.d.Perovskia abrotanoides NE D + n.d.Plectranthus ciliatus* NE M + –Salvia officinalis NE M + –Salvia splendens* NE M + –Satureja montana NE M + –Lamium album SC D – n.d.Lamium galeobdolon SC D – n.d.Lamium maculatum SC D – n.d.Lamium purpureum SC D – n.d.Leonurus cardiaca SC M – +Marrubium vulgare SC M – –Molucella laevis SC D – n.d.Phlomis russeliana SC D – n.d.Phlomis tuberosa SC M – +Stachys alpina SC D – n.d.

Oleaceae Olea europaea M – –Plantaginaceae Digitalis lanata M – –

Digitalis lutea M – –Plantago media M – +Plantago nivalis M – –Plantago schwarzenbergiana M – –Plantago sempervirens M – +

Scrophulariaceae Calceolaria scabiosifolia M – +Chelone lyonii M – +Linaria triornithophora M – –Nemesia strumosa M – –Penstemon digitalis M – +Penstemon hirsutus M – –Penstemon serrulatus M – –Scrophularia nodosa M – –Verbascum phlomoides M – –Verbascum undulatum M – –Veronica longifolia M – –

Verbenaceae Lantana camara M – –Verbena spec. M – –Verbena officinalis M – –Verbena rigida M – –Verbena urticifolia M – –

Order: SolanalesConvolvulaceae Convolvulus tricolor M – +Menyanthaceae Menyanthes trifoliata M – +Solanaceae Atropa belladonna M – +

Datura stramonium M – –Lycopersicon esculentum M – +Nicotiana sylvestris M – +Petunia hybrida M – n.d.Physalis alkekengi M – –Saracha edulis M – –Withania somnifera M – +

(continued on next page)


Table 2 (continued)


Order: ApialesApiaceae Anthriscus cerefolium M – +

Anthriscus sylvestris M – +Apium graveolens M – +Astrantia major * M + +Carum carvi M – +Cenolophium denudatum M – +Dorema ammoniacum M – +Eryngium bourgatii M – +Foeniculum vulgare M – +Levisticum officinale M – +Ligusticum scoticum M – –Peucedanum officinale M – –Sanicula europaea M + n.d.Sanicula marilandica M – –Seseli hippomarathrum M – –Seseli libanotis M – –

Araliaceae Aralia californica M – +Eleutherococcus senticosus M – +Hedera colchica M – +Hedera helix M – +

Order: AsteralesAsteraceae Achillea millefolium M – +

Arnica chamissonis M – +Centaurea macrocephala M – +Cichorium intybus M – +Helianthus annuus M – +Tagetes tenuifolia M – –

Order: DipsacalesCaprifoliaceae Diervilla lonicera M – +

Diervilla trifolia M – +Lonicera demissa M – +Lonicera emphyllocalyx M – +Lonicera ferdinandi M – +Lonicera kamtschatica M – +Lonicera syringantha M – –Viburnum dilatatum M – +Viburnum hupehense M – +Viburnum lantana M – +

Dipsacaceae Cephalaria gigantea M – –Dipsacus laciniatus M – +Knautia dipsacifolia M – +Scabiosa atropurpurea M – +Scabiosa caucasia M – +Succisella inflexa M – +

Uncertain order, Boraginales?Boraginaceae incl. Cerinthe major* M + –Hydrophyllaceae Echium italicum* M + +

Heliotropium amplexicaule* M + –Lindelofia longiflora* M + –Lithospermum arvense* M + –Lithospermum erythrorhizon - + n.d.Lithospermum purpureocoeruleum* D + n.d.Nonea lutea* M + +Symphytum asperum* M + –Symphytum officinale M + –Hydrophyllum canadense* M + +Hydrophyllum virginicum* M + –Nemophila menziesii* M + +Phacelia tanacetifolia* D + n.d.

Table 3Similarities/identities of amino acid sequences of H(P)PR from Coleus blumei (syn. Solenostemon scutellarioides, Lamiaceae) and similar data base entries.

Plant source, assigned function Amino acid sequence

Accession number Length Identity (%) Similarity (%)

Coleus blumei, HPPR 313 100 100Salvia miltiorrhiza, putative HPPR Q15KG6 313 90 97Salvia miltiorrhiza, putative HPPR A7KJR2 313 89 97Salvia miltiorrhiza, putative HPPR A9CBF7 313 89 96Vitis vinifera, putative uncharacterised protein A5CAL1 313 77 87Arabidopsis thaliana, putative D-isomer-specific 2-hydroxyacid dehydrogenase Q9CA90 313 76 87Populus jackii, putative uncharacterised protein A9PIN2 314 75 85



The transformation of phenylalanine is catalysed by the enzymesof the general phenylpropanoid pathway phenylalanine ammo-nia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H) and 4-couma-

Table 4Hydroxycinnamoyltransferases and their substrate preferences; pHPL = 4-hydroxyphenylla

Plant source (Acc. number) Donor substrate

Cynara cardunculus ‘‘HCT” [DQ104740] 4-coumaroyl-CoA, caffeoyl-CoANicotiana tabacum ‘‘HCT” [AJ507825] caffeoyl-CoA > 4-coumaroyl-CoA > feruloyl-

CoA > sinapoyl-CoANicotiana tabacum ‘‘HQT” [AJ582651] 4-coumaroyl-CoA, caffeoyl-CoALycopersicon esculentum ‘‘HQT”

[AJ582652]n.d.

Coffea canephora ‘‘HQT” [EF153930,EF153931]

n.d.

Coffea canephora ‘‘HCT” [EF137954,EF153929]

Hydrolysis of chlorogenic acid

Coffea arabica ‘‘HCT” [EF143341] n.d.Coleus blumei ‘‘HST” 4-coumaroyl-CoA, caffeoyl-CoA

Coleus blumei ‘‘RAS” [AM283092] 4-coumaroyl-CoA, caffeoyl-CoA

Melissa officinalis ‘‘RAS” 4-coumaroyl-CoA, caffeoyl-CoA

Plectranthus fruticosus ‘‘RAS” 4-coumaroyl-CoA, caffeoyl-CoA

Coleus forskohlii ‘‘RAS” 4-coumaroyl-CoA, caffeoyl-CoA

Fig. 3. Phylogenetic tree from 27 hydroxycinnamoyltransferase amino acid sequences w4.0 software package, bootstrapping with 500 replicates (Tamura et al., 2007). HRAS = rosmarinic acid synthase, HCBT = hydroxycinnamoyl/benzoyltransferase.

ric acid CoA-ligase (4CL). Tyrosine is transaminated by tyrosineaminotransferase (TAT) with 2-oxoglutarate as cosubstrate to 4-hydroxyphenylpyruvic acid (pHPP) which is then reduced to

ctic acid, DHPL = 3,4-dihydroxyphenyllactic acid, n.d. = not determined.

Acceptor substrate Reference

quinic > shikimic acid Comino et al. (2007)shikimic >> quinic acid Hoffmann et al. (2003)

quinic >> shikimic acid Niggeweg et al. (2004)n.d. Niggeweg et al. (2004)

n.d. Lepelley et al. (2007)

Lepelley et al. (2007)

n.d. Lepelley et al. (2007)shikimic >> quinic acid; not accepted:pHPL, DHPL

Sander and Petersen(unpublished)

pHPL, DHPL; not accepted: shikimic acid,quinic acid

Berger et al. (2006)


Weitzel and Petersen(unpublished)


Gehlen and Petersen(unpublished)


Petersen (unpublished)

ith assigned activities constructed with the neighbor joining program of the MEGACS/QT = hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase,


4-hydroxyphenyllactic acid (pHPL) by hydroxyphenylpyruvatereductase (HPPR). The two intermediary precursors are coupledby ester formation and with release of coenzyme A and 4-couma-royl-40-hydroxyphenyllactic acid (4C-pHPL) is formed. The reactionis catalysed by ‘‘rosmarinic acid synthase” (RAS; 4-coumaroyl-CoA:40-hydroxyphenyllactic acid 4-coumaroyltransferase). The 3-and 30-hydroxyl groups are finally introduced by cytochromeP450-dependent monooxygenase reactions. Looking at the eightenzymatic activities involved in this biosynthetic pathway, only amaximum of four of them might be specific for RA biosynthesis.The enzymes of the general phenylpropanoid pathway (PAL, C4H,4CL) are ubiquitous in land plants since they provide the precur-sors for the formation of supporting material like lignin andUV-protecting phenolic pigments. TAT is a primary enzyme as wellbecause it forms pHPP that is needed for the biosynthesis of toc-opherols and plastoquinones (Hess, 1993; Douce and Joyard,1996). A reaction similar to the stereospecific reduction of pHPPis active in photorespiration where hydroxypyruvate is reducedto glycerate by either a NADH-dependent peroxisomal hydroxy-pyruvate reductase (HPR) or a cytosolic NADPH-dependent HPR(Timm et al., 2008). Thus, HPPR might be identical or closely re-lated to this cytosolic HPR (see below), which is currently underinvestigation.

The remaining three enzymes, rosmarinic acid synthase (RAS)and the 3-and 30-hydroxylases, have been characterised in proteinand microsome preparations of suspension cells of C. blumei (Pet-ersen et al., 1993; Petersen, 1997). Recently, cDNAs encoding RASand a cytochrome P450 catalysing the 3- as well as the 30-hydrox-ylation (CYP98A14) have been cloned and heterologously ex-pressed (Berger et al., 2006; Eberle et al., 2009). Bothheterologously expressed proteins revealed a strict specificity forsubstrates involved in RA biosynthesis although they showed highsimilarities to 4-coumaroyl-CoA:quinate/shikimate hydroxy-cinnamoyltransferases and 4-coumaroylshikimate/quinate 3-hydroxylases, respectively (see below).

The biosynthesis of CA and caffeoylshikimic acid (CS) alsobranches off the general phenylpropanoid pathway (Fig. 1). 4-Cou-maroyl-CoA is the hydroxycinnamoyl donor for the esterification

Table 5Cytochrome P450 amino acid sequences of the CYP98A family and their assigned funcsimilarities are related to CYP98A14 from Solenostemon scutellarioides.

CYP98A14 Solenostemon scutellarioides 4C-DHPL 30-hydroxylase, Caf-pHPL 3-hydroxylaseCYP98A13v2 Ocimum basilicum, 4C-shikimate 3-hydroxylase, 4C-pHPL 3-hydroxylaseCYP98A13v1 Ocimum basilicum, 4C-shikimate 3-hydroxylase, 4C-pHPL 3-hydroxylaseCapsicum annuum, putative 4C 3-hydroxylase*

Vitis vinifera, cytochrome P450, n.f.a.Populus trichocarpa, coumaroyl 3-hydroxylase*

Populus alba x P. grandidentata, 4C-CoA 3-hydroxylaseCYP98A2 Glycine max, n.f.a.CYP98A46 Coptis japonica var. dissecta, n.f.a.CYP98A37 Medicago truncatula, n.f.a.CYP98A21 Ammi majus, n.f.a.CYP98A-C2 Coffea canephora, p-coumaroyl ester 3-hydroxylaseCYP98A3 Arabidopsis thaliana, 4-coumaroyl ester 3-hydroxylaseArabidopsis thaliana, cyt. P450-like protein, n.f.a.Camptotheca acuminata, cyt. P450, n.f.a.Coffea canephora, p-coumaroyl quinate/shikimate 3-hydroxylaseCYP98A-C1 Coffea canephora, putative p-coumaroyl 3-hydroxylasePinus taeda, p-coumarate 3-hydroxylaseCYP98A33v1 Nicotiana tabacum, n.f.a.CYP98A33v2 Nicotiana tabacum, n.f.a.Ginkgo biloba, 4-coumarate 3-hydroxylase*

Sesamum indicum, p-coumarate 3-hydroxylase*

CYP98A6 Lithospermum erythrorhizon, 4C-pHPL 3-hydroxylaseCYP98A1 Sorghum bicolor, cyt. P450

with quinic and shikimic acid. Hydroxycinnamoyl-CoA:shikimic/quinic acid hydroxycinnamoyltransferases have been detected onenzyme level (Stöckigt and Zenk, 1974; Ulbrich and Zenk, 1979,1980) and the respective genes characterised recently (Hoffmannet al., 2003; Niggeweg et al., 2004). An alternative pathway hasbeen detected in sweet potato (Ipomoea batatas, Convolvulaceae)where 4-coumaroylglucose serves as donor for the 4-coumaroylmoiety (Kojima and Villegas, 1984). The meta-hydroxylation ofthe 4-coumaroyl part of the ester is mediated by cytochromeP450 monooxygenases as described on enzyme level by Hellerand Kühnl (1985) and Kühnl et al. (1987). The first evidence for thishydroxylase on gene level was from Arabidopsis thaliana (Schochet al., 2001; Anterola et al., 2002; Franke et al., 2002; Nair et al.,2002). CA can be accumulated up to high levels as defense com-pound, e.g. in coffee, while CS was not found in high concentrationsin most plants (Sondheimer, 1964). In the current hypothesis CSmight serve as donor for caffeoyl moieties after re-transfer to coen-zyme A (Fig. 1).

3. D-isomer-specific 2-hydroxyacid dehydrogenases

The family of D-isomer-specific 2-hydroxyacid dehydrogenaseswas defined by Grant (1989). Members of this family catalyse theNAD(P)H-dependent reduction of 2-oxoacids to the correspondingD-isomers of 2-hydroxyacids as well as the reverse reaction. Mostknown members of this family are involved in primary metabolismand are predominantly from prokaryotic organisms, examples areD-lactate dehydrogenase (D-LDH), D-3-phosphoglycerate dehydro-genase or hydroxypyruvate reductase (HPR). The proteins are char-acterised by two domains, a substrate-binding or catalytic domainand a cosubstrate-binding domain. The majority of enzymes preferNADH as cosubstrate. The catalytic cleft is located between the twodomains. Active enzymes mostly are homodimers. In rare cases athird domain and a quarternary structure as homotetramer are ob-served, as for example for D-3-phosphoglycerate dehydrogenasefrom Escherichia coli (Schuller et al., 1995). It should be stressedthat enzymes catalysing the homologous reactions with L-isomersof the 2-hydroxyacids belong to different protein families. Several

tions; * = catalytic activity not shown, n.f.a. = no function assigned. The identities/

Amino acid sequence

Accession number Length Identity (%) Similarity (%)

Q84VU3 507 100 100Q8L5H7 509 80 90Q8L5H8 512 79 90B5LAT7 511 77 88A7PTC8 508 76 87B2Z6P3 508 77 87B0LXE6 508 77 87O48922 509 77 87A9ZT63 511 77 88Q2MJ10 509 75 86Q6QNI3 509 73 87Q2Q093 508 75 88Q940C7 508 74 85Q0WWH4 508 74 85Q5QIB0 509 71 85A4ZKM5 508 70 85Q2Q094 508 69 85Q8VZH6 512 70 83A1XEI4 508 69 84A1XEI5 520 69 84B1WAN6 512 70 83Q8VWQ9 509 66 81Q8GSQ6 506 65 81O48956 512 66 81


crystal structures of D-isomer-specific 2-hydroxyacid dehydrogen-ases have been solved, e.g. D-LDH from Lactobacillus helveticus(2dld, Bernard et al., 1995) or Lactobacillus bulgaricus (1j49, 1j4a,Razeto et al., 2002), HPR from Hyphomicrobium methylovorum(1gdh, Goldberg et al., 1994), human HPR (2gcg, Booth et al.,2006) and others. The crystal structure of HPPR from C. blumei issolved as well and will be published soon (Janiak et al., submitted).

3.1. Hydroxy(phenyl)pyruvate reductase from C. blumei

In RA biosynthesis, hydroxyphenylpyruvate reductase (HPPR)channels a primary metabolite, 4-hydroxyphenylpyruvate whichis also involved in the biosynthesis of tocopherols and/or plastoqui-nones (Hess, 1993; Douce and Joyard, 1996), into secondary metab-olism. The newly formed OH group is necessary for the followingesterification reaction. Based on peptide sequences of the purifiedprotein from suspension cultures of C. blumei a cDNA was clonedand the protein heterologously expressed. The protein catalysedthe NAD(P)H-dependent reduction of pHPP and 3,4-dihydroxyphe-nylpyruvate (DHPP) to the corresponding lactates (Fig. 1; Kim et al.,2004). Further biochemical studies, however, showed that theheterologously expressed protein could additionally reduce otheraromatic substrates (4-hydroxy-3-methoxyphenylpyruvate, phe-nylpyruvate) as well as small substrates such as hydroxypyruvate,pyruvate and glyoxylate. The highest affinity was shown forhydroxypyruvate which is an intermediate of photorespiration inplants. The photorespiratory hydroxypyruvate reductases (HPRs)exist as NADH-dependent peroxisomal and as NADPH-dependentcytosolic forms. From C. blumei the sequences of the peroxisomalHPR and the cytosolic HPPR show only 30% identity on amino acidlevel. Both proteins, however, belong to the family of D-isomer-spe-cific 2-hydroxyacid dehydrogenases. The amino acid sequence ofthe peroxisomal HPR shows the typical carboxyterminal peroxi-some targeting sequence (own unpublished results). The functionof the cytosolic HPR was unclear for a long time since the steps ofphotorespiration take place in chloroplasts, peroxisomes and mito-chondria. Recently it was shown that the cytosolic HPR can replacethe peroxisomal enzyme and thus was considered a bypass enzymein photorespiration (Timm et al., 2008).

The HPPR-cDNA from C. blumei (EMBL accession numberAJ507733) encodes a protein of 313 amino acid residues. Very sim-ilar cDNAs (EMBL accession numbers DQ099741, EF458148,DQ266514) have been cloned from S. miltiorrhiza, a Lamiaceae spe-cies also accumulating RA, (see Table 3) but the catalytic activitiesof the encoded proteins have not yet been shown. A cDNA encod-ing a protein with 87% similarity on amino acid level has beencloned from A. thaliana (accession number Q9CA90), which is notknown for RA accumulation.

It remains to be shown whether the NADPH-dependent cyto-solic HPR/HPPR is an enzyme involved in two different pathways,a primary one like photorespiration and a secondary metabolicpathway, namely RA biosynthesis. Alternatively a more specificHPPR has to be detected in RA-synthesising plants. Experimentsto clarify the role of H(P)PR in RA biosynthesis using the RNAi-ap-proach are currently done in our laboratory.

4. CoA-ester-dependent BAHD hydroxycinnamoyltransferases

4.1. Hydroxycinnamoyltransferases in phenolic metabolism

Many acyltransferases transfering the acyl moiety from a coen-zyme A thioester to an acceptor molecule in phenolic metabolismbelong to the superfamily of BAHD acyltransferases, which wasnamed after the four first known enzymes of this family (St. Pierreand De Luca, 2000; D’Auria, 2006). The transfer of acyl moieties

from glucose esters in plant secondary metabolism is catalysedby a different protein family, namely members of the serine car-boxypeptidase-like acyltransferase family (Milkowski and Strack,2004). The A. thaliana and Oryza sativa genomes revealed 64 and119 members of the BADH superfamily, respectively. Conservedsequence motifs of his BAHD acyltransferase family are theHxxxD(G) motif directly involved in substrate binding and theDFGWG motif located more to the C-terminal end. D’Auria (2006)has defined five different clades of BAHD acyltransferases: clade Iconsists mostly of anthocyanin-modifying acyltransferases, herea third typical motif (YFGNC) can be used for classification. CladeII is not very well defined but includes members of epicuticularwax biosynthesis. Clade III comprises alcohol acetyltransferasesand acyltransferases involved in alkaloid biosynthesis. Clade IV in-cludes agmatine N-hydroxycinnamoyltransferase. In clade V, sub-groups catalysing the formation of volatile compounds (e.g.benzylbenzoate), alkaloids and a subgroup comprising hydroxycin-namoyl/benzoyltransferases can be defined. In this last subgroup,hydroxycinnamoyltransferases involved in the biosynthesis ofhydroxycinnamoyl esters such as 4-coumaroylshikimate or -qui-nate or CA are found. According to St. Pierre and De Luca (2000)the evolutionary origin of the BAHD acyltransferase superfamilymight lie in the CAT (chloramphenicol acetyltransferase) gene fam-ily. The known sequences from plants indicate a monophyletic ori-gin of all plant genes of this family.

Crystal structures of members of the BAHD superfamily havebeen solved for vinorine synthase from Rauvolfia serpentina (pdb-code: 2bgh; Ma et al., 2005), anthocyanin malonyltransferase fromDendranthema morifolium (pdb-codes: 2e1t, 2e1u, 2e1v; Unnoet al., 2007) and trichothecene 3-O-acetyltransferase from Fusar-ium sporotrichioides and Fusarium graminearum (pdb-codes: 2rkv,2rkt, 3b2s, 3b30, 2zba; Garvey et al., 2008).

Different hydroxycinnamoyltransferases of the BAHD superfam-ily are involved in the biosynthesis of phenolic compounds.N-Hydroxycinnamoyltransferases such as agmatine N-hydroxycin-namoyltransferase (Burhenne et al., 2003) or (hydroxy)anthranilateN-hydroxycinnamoyltransferase (Yang et al., 1997, 2004) and flavo-noid glycoside/anthocyanin hydroxycinnamoyltransferases (asreviewed by Nakayama et al. (2003)) will not be discussed here.The hydroxycinnamoyltransferases discussed here show a moder-ate specificity for their hydroxycinnamoyl donors accepting both4-coumaroyl- and caffeoyl-CoA and often also other (hydroxy)cin-namic acid CoA-thioesters but sometimes have more distinct spec-ificities for their acceptor substrates (Table 4). Recent investigationssuggested that predominantly CS and to a lesser extent CA (at leastin some plants) are the precursors for caffeic acid and/or caffeoyl-CoA which will be incorporated into phenolic compounds or trans-formed to lignin monomers with more elaborated substitution pat-terns at the aromatic ring (3-methoxy-4-hydroxy, 3,5-dimethoxy-4-hydroxy). This renders hydroxycinnamoyl-CoA:quinate/shikim-ate hydroxycinnamoyltransferases (HCS/QT) and/or hydroxycinna-moyl-CoA:shikimate hydroxycinnamoyltransferases (HST) centralenzymes of phenolic metabolism (Fig. 1; Schoch et al., 2001; Hoff-mann et al., 2003). The enzymes forming caffeoyl-CoA by transferingthe 4-coumaric acid moiety from 4-coumaroyl-CoA to shikimateand/or quinate (HCS/QT, HST, HQT) and the 3-hydroxylases of theCYP98A family introducing the 3-OH group into the aromatic ring(see below) thus should be essential for the formation of e.g. caffeicacid derivatives and lignin monomers. Consequently, silencing ofHCS/QT in Nicotiana benthamiana and A. thaliana affected lignin for-mation with respect to amount and composition (Hoffmann et al.,2004, 2005). Similarly, lignin formation in Pinus taeda or Medicagosativa was affected by silencing HST (Wagner et al., 2007; Shadleet al., 2007).

Hydroxycinnamoyltransferases only or preferably using quinateas acceptor substrate (HQT) are essential for the biosynthesis of CA,


the most ubiquitously occurring phenolic ester in the plant king-dom. Down-regulation or silencing of HQT in tomato affected thelevels of CA while lignin formation was not affected. Up-regulationresulted in enhanced CA levels and increased antioxidative capacityand pathogen resistance (Niggeweg et al., 2004). Furthermore, theplant’s UV-protection was dependent on HQT expression and onthe accumulation of CA (Clé et al., 2008). The differing expression

Fig. 4. Phylogenetic tree from 36 amino acid sequences of members of the CYP98A famil(500 replicates) of the MEGA 4.0 program package (Tamura et al., 2007) were used. TAF022458, CYP98A3: O22203, CYP98A4: Q65X81, CYP98A5: AY963109, CYP98A6: ABAJ583530, CYP98A11: AJ583531, CYP98A12: AJ583532, CYP98A13v1: AY082611, CYP98CYP98A19: AY064170, CYP98A20: AY065995, CYP98A21: AY532371, CYP98A23/24/2constructed from Zea mays ESTs, CYP98A34: constructed from Physcomitrella patens ESDN838382 (cDNA), CYP98A39v1: AJ585988, CYP98A39v2: AJ585990, CYP98A39v3: AJ5Ginkgo biloba C3H: B1WAN6, CYP73A5: AAC99993.

patterns of HCS/QT and HQT in Coffea canephora were correlatedto lignin and CA formation, respectively (Lepelley et al., 2007).

The number of HCS/QTs and the relative use of quinic or shiki-mic acid as substrate (Table 4) differ in plant species. While in A.thaliana the absence of a CA-forming enzyme is consistent withthe absence of hydroxycinnamoylquinates, six hydroxycinnamoyl-transferase sequences were detected in Populus tremuloides which

y; CYP73A5 was used as outgroup. The neighbor joining and bootstrapping featureshe accession numbers of the used sequences are: CYP98A1: AF029856, CYP98A2:017418, CYP98A7: AY107051, CYP98A8: Q9CA61, CYP98A9: Q9CA60, CYP98A10:A13v2: AY082611, CYP98A14: AJ427452, CYP98A18: Oryza sativa genome project,5v1/27: Populus trichocarpa genome project, CYP98A28: AY466856, CYP98A29:Ts, CYP98A35: DQ269126, CYP98A36: DQ269127, CYP98A37: Q2MJ10, CYP98A38:85991, CYP98A40: AJ585989, CYP98A43: AM435080 (gDNA), CYP98A46: A9ZT63,


accumulates an abundance of hydroxycinnamoyl esters (Tsai et al.,2006).

The third type of hydroxycinnamoyltransferase in this contextis hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinna-moyltransferase (rosmarinic acid synthase, RAS) which useshydroxyphenyllactic acids but not quinate or shikimate as acceptorsubstrates. The first cDNA for RAS was cloned from C. blumei (syn.Solenostemon scutellarioides), a RA-accumulating Lamiaceae species(Berger et al., 2006). The properties of this enzyme will be pre-sented below.

4.2. Properties of hydroxycinnamoyl-CoA:hydroxyphenyllactatehydroxycinnamoyltransferase (rosmarinic acid synthase; RAS)

The specific enzyme of RA biosynthesis is the hydroxycinna-moyltransferase that transfers the hydroxycinnamic acid moietyfrom hydroxycinnamoyl-CoA to the aliphatic hydroxyl group of ahydroxyphenyllactate thus forming an ester. This enzyme has beendescribed from suspension cultures of C. blumei as rosmarinic acidsynthase (RAS; Petersen and Alfermann, 1988; Petersen, 1991; Pet-ersen et al., 1993). The substrate affinities of RAS indicated thepreferential acceptance of monohydroxylated substrates, 4-cou-maroyl-CoA and pHPL. The finalisation of the rosmarinic acid sub-stitution pattern thus would be the final steps of RA biosynthesis,catalysed by 3- and 30-hydroxylases (Petersen, 1997), as describedbelow. Caffeoyl-CoA and 3,4-dihydroxyphenyllactate are acceptedas RAS substrates as well.

The cDNA for RAS (EMBL accession number AM283092) wascloned using peptide information from the purified RAS proteinisolated from suspension cells of C. blumei (Berger et al., 2006).The cDNA has an open reading frame of 1290 base pairs encodinga protein of 430 amino acid residues with a calculated molecularmass of 47.9 kDa with a theoretical isoelectric point of 5.89. Thegenomic sequence revealed a single phase 0 intron of 914 basepairs after 405 nucleotides. According to St. Pierre and De Luca(2000) this is a typical ‘‘Q-intron” (inserted after the conservedamino acid Q = glutamine) 17 amino acid residues forward of theconserved motif HxxxDG. This intron is highly conserved in theBADH superfamily of acyltransferases and this was seen as consis-tent with the hypothesis that the plant sequences are derived fromone common ancestor.

The His-tagged RAS protein was expressed in E. coli and purifiedby metal chelate chromatography. The enzyme accepted 4-couma-royl-CoA and caffeoyl-CoA as hydroxycinnamoyl donors with veryhigh affinities. 4-Hydroxyphenyllactate and 3,4-dihydroxyphenyl-lactate served as acceptors while shikimate and quinate were noacceptor substrates. Despite the high sequence similarities (see be-low) between RAS and HCS/QTs these enzymes have evolved diver-gently with respect to their substrate binding centres. Thesubstrate specificities of hydroxycinnamoyltransferases differ con-siderably (Table 4). While some hydroxycinnamoyltransferasesprefer quinic over shikimic acid, others strongly prefer shikimicacid or only use this acceptor. The enzymes characterised to date

Fig. 5. 3D structures of the acceptor substrates of hydroxycinnamoyltransferases. Th

to accept hydroxyphenyllactates are extremely specific for thesesubstrates. RAS and HCS/QT from C. blumei, which preferentiallyaccepts shikimate as acceptor molecule, have identities/similaritiesof 56/73% on amino acid level and 66% on nucleic acid level. Inter-estingly, especially the region adjoining the conserved amino acidsHxxxDG shows differing amino acids for RAS and for HCS/QT. Asimilar specificity with respect to the acceptor molecules shikimicacid and pHPL acid has been reported for two partially purified 4-coumaroyltransferases from Ocimum basilicum (Lamiaceae; Ganget al., 2002). The enzyme accepting shikimate as substrate washighly expressed in peltate glandular trichomes synthesising euge-nol but not in those accumulating (methyl)chavicol without ameta-hydroxy group. This can be taken as an indication of theimportance of the formation of 4-coumaroyl esters for the estab-lishment of the 3,4-dihydroxy substitution pattern also for com-pounds other than monolignols.

A phylogenetic tree (neighbor joining and bootstrapping fea-tures (500 replicates) of the MEGA 4.0 program package; Tamuraet al., 2007) constructed from 27 sequences of BAHD hydroxy-cinnamoyltransferases with assigned catalytic affinities showeddistinct groups (Fig. 3). Hydroxycinnamoyltransferases usinganthocyanins as acceptor substrates group together, another groupis formed by hydroxycinnamoyl/benzoyltransferases (HCBTs). En-zymes using shikimate and/or quinate are clearly separated fromhydroxycinnamoyltransferases with hydroxyphenyllactates assubstrate. Currently several more hydroxycinnamoyltransferasesequences putatively involved in RA biosynthesis are being inves-tigated and will complete our view in near future. Modellingand/or crystallisation studies, which are currently done in our lab-oratory, will show which differences in the architecture of the ac-tive centers of 4-coumaroyl-CoA:4-hydroxyphenyllactate andshikimate/quinate hydroxycinnamoyltransferases explain the dif-ferent substrate acceptances.

5. Cytochrome P450 CYP98A

5.1. CYP98A in phenylpropanoid metabolism

Three families of cytochrome P450s have been described to beactive in basic phenylpropanoid metabolism, namely CYP73 withcinnamic acid 4-hydroxylase (C4H), CYP84 with coniferaldehyde5-hydroxylase (CA5H) and CYP98 whose members with knownactivities are aromatic meta-hydroxylases (Ehlting et al., 2006).The CYP73 and CYP98 families may share a common ancestor(Schoch et al., 2001). Generally, the CYP98A family is to dateknown to introduce the 3-OH group into the 4-coumaroyl moietiesof hydroxycinnamic acid esters or amides such as 4-coumaroylqu-inate or 4-coumaroylshikimate or to a lesser extent 4-coumaroyl-tyramine (Ehlting et al., 2006; Morant et al., 2007). The substratepreference usually is 4-coumaroylshikimate over 4-coumaroylqui-nate while 4-coumaroyltyramine is mostly not accepted with ahigh affinity (Ehlting et al., 2006). One of the major knownfunctions of CYP98A monooxygenases � in cooperation with

e OH group that accepts the 4-coumaric acid moiety is indicated by an arrow.


hydroxy-cinnamoyltransferases – is the establishment of the caf-feic acid substitution pattern of phenylpropanoid moieties, puta-tively via 4-coumaroylshikimic acid (Schoch et al., 2001; Anterolaet al., 2002; Franke et al., 2002; Nair et al., 2002), and the biosyn-thesis of CA via 4-coumaroylquinic acid (Niggeweg et al., 2004).More than 46 partial or complete CYP98A sequences are known(cytochrome P450 database by Dr. David Nelson), but most of themhave not been expressed and tested for their catalytic activities.Since the involvement in the formation of lignin precursors ofCYP98A3 is known and is anticipated for others, CYP98 sequenceshave to be present in virtually all higher plant (tracheophyte) gen-omes. In wheat, duplication events resulted in eight CYP98A se-quences three of which showed meta-hydroxylation activity of 4-coumaroyl moieties after heterologous expression (Morant et al.,2007). More than one sequence have also been deposited for e.g.Populus trichocarpa (CYP98A23, CYP98A24, CYP98A25, CYP98A26,CYP98A27), C. canephora (CYP98A35, CYP98A36), O. sativa(CYP98A4, CYP98A18) and A. thaliana (CYP98A3, CYP98A8,CYP98A9).

5.2. Properties of 4-coumaroyl-4’-hydroxyphenyllactate 3- and 3’-hydroxylase from C. blumei (CYP98A14)

In RA biosynthesis in C. blumei, cytochrome P450-dependentmonooxygenases are involved in the formation of 4-coumaric acidfrom t-cinnamic acid (C4H) and in the 3- and 30-hydroxylation of4-coumaroyl-30,40-dihydroxyphenyllactate (4C-DHPL) and caf-feoyl-40-hydroxyphenyllactate (Caf-pHPL), respectively, or 4-cou-maroyl-4’-hydroxyphenyllactate (4C-pHPL) finalising the RAmolecule (Petersen, 1997). Experiments with microsomal prepara-tions from suspension-cultured cells of C. blumei suggested theinvolvement of two different cytochrome P450s in the introduc-tion of the 3- and 30-hydroxyl groups (Petersen, 1997). We re-cently isolated the cDNA and genomic DNA of a cytochromeP450 from this species which was assigned the number CYP98A14(Nelson, personal communication). The cDNA was expressed inSaccharomyces cerevisiae and characterised with respect to itscatalytic activity (Eberle et al., 2009). The main activity is the 3-hydroxylation of 4C-DHPL and to a lesser extent the 3’-hydroxyl-ation of Caf-pHPL while 4-coumaroylshikimate or -quinate werenot hydroxylated. CYP98A6 from L. erythrorhizon (Matsuno et al.,2002) and CYP98A13 from O. basilicum (Gang et al., 2002) – bothplant species that are known for their accumulation of RA – werereported to accept 4C-pHPL as substrate and to hydroxylate in po-sition 3 of the 4-coumaroyl moiety, although to a low extent(CYP98A13) compared to 4-coumaroylshikimate as substrate.Most members of the CYP98A family have not yet been testedwith respect to their catalytic activities. CYP98A14 from C. blumeidiffers from the other characterised CYP98As since it does not ac-cept 4-coumaroylquinate or -shikimate as substrate and it is addi-tionally able to hydroxylate both aromatic rings of 4C-DHPL andCaf-pHPL, respectively, thus acting as 3-hydroxylase as well asas 30-hydroxylase (Eberle et al., 2009). The apparent Km-values,however, differ with 5 lM for 4C-DHPL and 40 lM for Caf-pHPL,showing that the 3-hydroxylation is the preferred reaction. Thisleaves the possibility open that a yet unknown CYP gene mightencode a specific 30-hydroxylase in RA biosynthesis. A similar sit-uation has recently been described for Sesamum indicum, whereexperiments with microsomal preparations suggested that twocytochrome P450s are involved in the formation of the two meth-ylenedioxy bridges of the sesamin molecule from pinoresinol (Jiaoet al., 1998), while the heterologously expressed protein encodedby the CYP81Q cDNA was able to form both methylenedioxybridges (Ono et al., 2006). CYP98A14 from C. blumei (EMBL acces-sion number AJ427452; Eberle et al., 2009) showed highest se-quence identities with 74% on nucleotide level and 80% on

amino acid level to CYP98A13v2 from O. basilicum (EMBL acces-sion number AY082612; Gang et al., 2002) while the identitiesto CYP98A6 from L. erythrorhizon (EMBL accession numberAB017418; Matsuno et al., 2002) ranged at 68% and 65%, respec-tively (Table 5). The cDNA comprised an open reading frame of1521 bp encoding a protein of 507 amino acid residues with a cal-culated molecular mass of 57.6 kDa. The genomic DNA sequenceof CYP98A14 from C. blumei revealed two introns: the first oneis a phase 1 intron starting after 487 nucleotides with a lengthof 81 bp, the second phase 0 intron of 95 base pairs starts after876 nucleotides of the coding sequence (Wolters and Petersen,unpublished). With this respect the genomic structure is similarto CYP98A35 from C. canephora which also has two introns, aphase 1 intron (116 bp) after 484 nucleotides and a phase 0(106 bp) intron after 882 nucleotides (Mahesh et al., 2007). Thegenomic sequence of a ‘‘p-coumarate 3-hydroxylase” from Ginkgobiloba, an ancient gymnosperm, also had two introns after 487and 883 nucleotides of the cDNA sequence with lengths of 693and 383 base pairs (Liu et al., 2008). The second intron was re-ported to be conserved in CYP genes of the A-clade (Paquetteet al., 2000), while the first intron is not always present.

A phylogenetic tree (neighbor joining and bootstrapping fea-tures (500 replicates) of the MEGA 4.0 program package; Tamuraet al., 2007) was constructed on the basis of 36 full-length aminoacid sequences of members of the CYP98A family (Fig. 4); C4H fromA. thaliana (CYP73A5) was used as outgroup. The phylogram clearlyshows groupings of CYP98As from lower plants (Selaginella moel-lendorffii, Physcomitrella patens), gymnosperms (P. taeda, G. biloba)and monocotyledonous angiosperms. Three sequences of CYP98Asfrom two species (O. basilicum, C. blumei) accepting precursors ofRA are grouped together while CYP98A6 from L. erythrorhizonwhich was reported to accept 4C-pHPL (other substrates havenot been tested) groups with a 4-coumaroyl-CoA:shikimate/qui-nate 3-hydroxylase from C. canephora suggesting that a further elu-cidation of the substrate specificities of CYP98A6 should beperformed.

The high sequence similarities between CYP98As acting in RAbiosynthesis and in CA/CS biosynthesis as well as the substratepromiscuity of some of the CYP98As accepting both, substratesfor CA/CS and RA biosynthesis, makes an evolutionary relationshipprobable. Our current knowledge with DNA sequences only avail-able from members of two higher plant families, Lamiaceae andBoraginaceae, makes it impossible to know whether the recruit-ment of CYP98A from lignin monomer or CA biosynthesis for RAbiosynthesis has taken place once or several times duringevolution.

6. Possible evolutionary relationship of caffeoylshikimic/chlorogenic acid and rosmarinic acid biosynthesis

With respect to their biosynthetic enzymes, rosmarinic acidand CA/CS biosynthesis seem to be rather similar and evolution-arily related. In both cases 4-coumaroyl-CoA coming from thegeneral phenylpropanoid pathway is used to transfer the 4-cou-maroyl moiety to the OH group of an acceptor molecule: shiki-mic, quinic or hydroxyphenyllactic acid. The acceptor OHgroups differ considerably: the hydroxyl group in hydroxyphe-nyllactic acid is in the side chain which should be rather flexible,while in shikimic and quinic acid it is attached to closed rings.The distances between the carboxyl groups of the acceptor acidsand the OH groups that are esterified also differ between quinicand shikimic acid on the one side and 4-hydroxyphenyllacticacid on the other (Fig. 5). Thus the active centers should havedifferent architectures which is currently under investigation inour laboratory.


Given that the formation of 4-coumaroylshikimate and itshydroxylation to CS are inherent steps of monolignol formationas proposed by Schoch et al. (2001), the enzymes catalysing thesesteps must have evolved at the latest in early land plants. Theoccurrence of monolignol-derived lignans and caffeic acid estershas been shown in ‘‘mosses”: a number of caffeic acid-derived lign-ans have been isolated e.g. from the liverworts Jamesoniellaautumnalis, Scapania undulata, Pellia epiphylla and Bazzania trilobata(Tazaki et al., 1995 and cited literature; Martini et al., 1998; gen-eral review: Asakawa, 2004). Anthocerotonic, megacerotonic androsmarinic acids have been detected in members of the hornworts(Takeda et al., 1990). Liverworts and hornworts are considered thefirst groups of land plants following ancestors from the Charophy-ceae green algae family. While lignin has not yet been reportedfrom these groups of land plants it appears in tracheophytes(Waters, 2003). Nevertheless, the capacity to synthesise cinnamicacid derivatives with a higher substituted aromatic ring (e.g. caffeicacid) is already present in the earliest land plant groups. Recentfindings demonstrate the presence of lignified cell walls with 4-coumaryl, coniferyl and sinapyl moieties from red algae (Martoneet al., 2009) and thus opens up the discussion about the timepointof the evolution of lignin biosynthesis and a mono- or polyphyleticorigin.

Peter and Neale (2004) postulated that gymnosperms andangiosperms share a common ancient and conserved pathway forthe formation of guiacyl lignin while the formation of syringyl lig-nin is a more recent achievement of angiosperms. The ancientpathway comprises the enzymes of the general phenylpropanoidpathway (PAL, C4H and 4CL), 4-coumaroyl-CoA:shikimatehydroxycinnamoyltransferase, 4-coumaroylshikimate 3-hydroxy-lase as well as caffeoyl-CoA 3-O-methyltransferase, (hydroxy)cin-namyl alcohol reductase (CCR) and (hydroxy)cinnamyl alcoholdehydrogenase (CAD) and leads to the formation of 4-coumarylalcohol and coniferyl alcohol as monolignols. Lignin formed fromthese monolignols is primarily found in water-conducting ele-ments. In angiosperms syringyl lignin contains a further monolig-nol, sinapyl alcohol, which needs further enzymes for itsbiosynthesis, namely coniferaldehyde 5-hydroxylase, caffeic acid/5-hydroxyconiferaldehyde 3/5-O-methyltransferase and a specificsinapyl alcohol dehydrogenase (SAD). Syringyl lignin is predomi-nantly formed in fiber cell walls and seems to be a more recentevolutionary achievement. This shows again that hydroxy-cinnamoyltransferases and hydroxycinnamoyl meta-hydroxylasesseem to be evolutionarily old enzymes. Alternatively the first landplants could have developed a differing set of enzymes to synthe-sise the caffeic acid and further derived substitution patterns inphenolic metabolites.

The biosynthesis of RA does not require many enzymes spe-cific for this biosynthetic pathway. As indicated above, the forma-tion of 4-coumaroyl-CoA should be an inherent metabolic featureof all land plants. Furthermore, the formation of pHPP is neces-sary for all photosynthetically active organisms since it is a pre-cursor for tocopherols and/or plastoquinones. Thetransformation of pHPP to pHPL could possibly be catalysed byan enzyme of photorespiration, the cytosolic HPR, or by an HPPRenzyme very closely related to HPR. There are high similaritiesbetween cytosolic HPRs and the putative HPPR from C. blumei.The transfer of the 4-coumaroyl moiety to pHPL and the introduc-tion of the 3,4-dihydroxy substitution patterns of both aromaticrings can be mediated by a hydroxycinnamoyltransferase and aaromatic meta-hydroxylase (CYP98A), which are very closely re-lated to enzymes in monolignol formation. Thus the achievementto synthesise RA could be very old which would be supported bythe fact that RA was already found in hornworts. Whether the en-zymes of hydroxycinnamoyl transfer and meta-hydroxylation inRA biosynthesis have evolved several times independently in dif-

ferent plant groups still remains open and will be the focus of ourfuture investigations.

Acknowledgement

The financial support by the Deutsche Forschungsgemeinschaft(DFG) in parts of this project is gratefully acknowledged.

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