Tropane and Granatane Alkaloid Biosynthesis

25 Pages·2016·3.33 MB·English

molecules
Review
Tropane and Granatane Alkaloid Biosynthesis:
A Systematic Analysis
NeillKim1,†,OlgaEstrada1,†,BenjaminChavez1,CharlesStewartJr.2andJohnC.D’Auria1,*
1 DepartmentofChemistryandBiochemistry,TexasTechUniversity,Lubbock,TX79409-1061,USA;
[email protected](N.K.);[email protected](O.E.);[email protected](B.C.)
2 OfficeofBiotechnology,IowaStateUniversity,Ames,IA50011-1079,USA;[email protected]
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
† Theseauthorscontributedequallytothisarticle.
AcademicEditor:MichaelWink
Received:30September2016;Accepted:7November2016;Published:11November2016
Abstract: Thetropaneandgranatanealkaloidsbelongtothelargerpyrrolineandpiperidineclasses
of plant alkaloids, respectively. Their core structures share common moieties and their scattered
distributionamongangiospermssuggestthattheirbiosynthesismaysharecommonancestryinsome
orders,whiletheymaybeindependentlyderivedinothers. Tropaneandgranatanealkaloiddiversity
arisesfromthemyriadmodificationsoccurringtotheircoreringstructures. Throughoutmuchof
humanhistory,humanshavecultivatedtropane-andgranatane-producingplantsfortheirmedicinal
properties. This manuscript will discuss the diversity of their biological and ecological roles as
wellaswhatisknownaboutthestructuralgenesandenzymesresponsiblefortheirbiosynthesis.
In addition, modern approaches to producing some pharmaceutically important tropanes via
metabolicengineeringendeavorsarediscussed.
Keywords: tropanealkaloids;granatanealkaloids;secondarymetabolism;metabolicengineering
1. Introduction
Plantsaresessileorganismsandthusevolvednaturalproductsor“specializedmetabolites”as
a chemical response to both biotic and abiotic forces. Specialized metabolites are used by plants
to defend themselves and communicate with other plants and organisms in their environments.
Whilstthechemicaldiversityofplantspecializedmetabolitesisvast,withtotalnumbersthoughtto
exceedover200,000structures,commonthemesofstructureandfunctionaretheresultofrepeated
andconvergentevolutionofboththeirbiosynthesisandbiologicalroles[1]. Moreover,thechemical
structures and underlying biosynthetic enzymes of specialized metabolites serve as inspiration to
medicinalandnaturalproductchemists.
Manyspecializedmetabolitesarepharmacologicallyactiveandhavebeenusedbyhumansfor
therapeuticandrecreationalpurposessincethebeginningofrecordedhistory. Inparticularalkaloids,
pharmacologicallyactivecyclicnitrogencontainingmetabolitesderivedfromaminoacids,areknown
fortheirpharmacologicaleffectsandfrequentlyserveasthestartingpointfordrugdevelopment[2].
Someoftheoldestdomesticatedmedicinalplantshavebeenthosethatproducealkaloids. Forexample,
Erythroxylumcoca,aspeciesnotablefortheproductionofthetropanealkaloidcocaine(1),wasusedin
Peruvianhouseholdsatleast8000yearsago[3]. Similarly,pomegranate(Punicagranatum)hasahistory
ofcultivationthatgoesbackatleast10,000yearsinEgyptandiswell-knownfortheproductionof
granatanealkaloids[4].
Tropane(TA)andgranatane(GA)alkaloidsarestructuralhomologues,sharingsimilarchemical
compositionsandcorescaffolds. Despitetheirsimilarities,TAsandGAsshowdifferentdistribution
patternsacrosstheplantkingdom. TheN-methyl-8-azabicyclo[3.2.1]-octanecorestructureofTAsis
Molecules2016,21,1510;doi:10.3390/molecules21111510 www.mdpi.com/journal/molecules
Molecules2016,21,1510 2of25
Molecules 2016, 21, 1510 2 of 24
ffoouunndd iinn oovveerr 220000 aalklkaaloloidids s[2[,25,,56,]6 (]F(iFgiugruer 1e).1 I)n. Icnonctornastrt,a Nst-,mNe-tmhyelt-h9y-al-z9a-baizcaybcilcoy[3c.l3o.[13].-3n.o1n]-annoen, athnee ,cothree
sccoarfefosclda ffoofl dGoAfsG, Aapsp,aeparpse ainrs cionncsoidnseirdaebrlayb fleywfeewr earlkaalkloaildo idmmetaetbaobloitleitse s(F(iFgiugruer e11).) .TThhee bbiiccyycclliicc ccoorree
ssttrruuccttuurreess ooff TTAAss aanndd GGAAss ddiiffffeerr bbyy oonnllyy oonnee ccaarrbboonn aattoomm,, yyeett tthhiiss ddiiffffeerreennccee aalltteerrss tthhee ccoonnffoorrmmaattiioonnaall
pprreeffeerreenncceess ooff eeaacchh ooff tthhee ccoorree sskkeelleettoonnss [[77]].. FFuurrtthheerrmmoorree,, tthhee pprreesseennccee oorr aabbsseennccee ooff aa ssiinnggllee ccaarrbboonn
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FFiigguurree 11.. ((aa)) TThhee ttrrooppaannee ccoorree sskkeelleettoonn;; ((bb)) TThhee bbiiccyycclliicc ggrraannaattaannee ccoorree sskkeelleettoonn.. BBootthh ssttrruuccttuurreess ddeeppiicctt
tthheeiirr cchheemmiiccaallllyy aacccceepptteedd ccaarrbboonn nnuummbbeerriinngg..
11..11.. SSiimmiillaarriittiieess aanndd DDiiffffeerreenncceess iinn MMeeddiicciinnaall PPrrooppeerrttiieess
DDeessppiittee tthheeiirr ssttrruuccttuurraall ssiimmiillaarriittiieess TTAAss aanndd GGAAss hhaavvee ddiissttiinncctt mmeeddiicciinnaall pprrooppeerrttiieess.. TTAAss hhaavvee
bbeeeenn ccoonnssiiddeerreedd ppaannaacceeaass tthhrroouugghhoouutt rreeccoorrddeedd hhiissttoorryy,, eessppeecciiaallllyy bbeeccaauussee ooff tthheeiirr aannttiicchhoolliinneerrggiicc
pprrooppeerrttiieess. .AnAtincthicohlionleirngeircgs iacrse aa rcelaass colfa csosmopfocuonmdps ouusendd ass udsreudgsa tso bdlroucgks thteo abctlioocnk otfh theea accteiotynlcohfoltihnee
naceeutryoltcrhaonlsimneittneeru troo ttrreaants mmoitttieorn tsoicktrneeasts manodti odnisesaisceksn seusscha ansd Adlzisheeaismeesr’ssu acnhda PsaArkliznhseoinm’se r[8’s]. aTnhde
mPaertkhiynlsaotend’s n[i8t]r.ogTehne inm eththe ycloarteed rinnigtr oofg ecnocianinteh e(1c)o arnedr inotgheorf TcoAcsa isneerv(e1s) aasn da osttrhuecrtuTrAals asneravloegs aosf
aacsettryulcchtuorlianlea. nTaAlos ghaovfea bceeteynl cohbosleirnvee.dT tAo sathtaacvhe tboe aenndo ibnsheirbvite dmutoscaatrtiancihc atoceatynldchionlhinibe irtemceupstocarsr i[n9i]c.
TacAesty folcuhnodli nine trheec eSpotlaonrsac[9ea].eT aAres wfoeulln kdnionwtnh efoSro blaontha ctheaeeir aarnetiwcheollliknneorgwicn afnodr baontthispthaesimraondtiicc phroolipneerrtgieisc
tahnadt aanffteicstp tahsem poadriacspymroppaetrhtieetsict hnaetrvaoffuesc tstyhsetepma r[a1s0y–m12p]a. tThheetsice npelarvnotsu hsasvyes tbeemen[1 u0s–e1d2 ]f.oTr hpeasien prlealinetfs,
ahnavesethbeeesina,u asnedd faosr ap tarienatrmelieenf,t afonre sdtrhuegsi aa,dadnicdtiaosna [t1r0e]a. tDmaetnutrafoer Fdlorus,g thade ddirciteiodn fl[o1w0]e.rDs aotfu DraaetuFrlao sm,ethteel
adlrsioe dknfloowwne rass o“fyDanagtujirnahmueat”e lina lCsohiknnao, whans abse“enya untgiljiiznehdu aan”din reCchorindae,dh ians tbheee Cnhuintieliszee Pdhaanrdmraeccooprodeeida
ains atnh eanCehstihneetsiec aPnhda rwmaas cporpeosceriiabeads taon traenate sctohuegthic, aasnthdmwaa asnpdr ceosncrviublesdiontos [t1r3e]a.t Pcrozeuwgahlk,iaa sttahnmguaticaan ids
aco rnavreu mlsieodnisci[n1a3l] .sPolraznewacaelkoiuast apnlgauntti cfoauisnadr ianr ethme eTdibiceitnaanl sPolalatenaauce oofu Cshpilnaan tinfo wunhdichin ththee rToiobtest, asneePdlas taenadu
eonftCirhei vneagientawtihviec htistshueerso aortes ,usteileizdesda [n1d4]e. nPt.i rtaenvgeugtiectaa ctiovnetatiisnssu seesvaerrealu btiiolilzoegdic[a1l4ly]. aPc.ttivane gTuAtisc ainccolnutdaiinngs
asenviseordaalmbiionleo g(2ic),a slclyopaocltaivmeinTeA (s3)in acnldu daitnrogpainneis (othdea mraicneem(i2c) m,sicxotuproel aomf hinyeos(c3y)aamndinaet (r4o)p. Tinhee( TthAesr parceesmenict
imn iPxt. utarnegouftihcay oasrecy aasmsoicnieat(e4d) .wTithhe mTaAnsy pbrieosleongticianl aPc.ttiavnitgieusti icnacalurediansgs oacniaaltgeedsiwc,i tshpamsman myobdioulloagtiiocnal,
pacetsitvicitiideasl,i nacnldu dainntgi-iannflaalmgemsiact,osryp aesfmfecmts o[d14u]l.a Htioynos,cypaemstuicsi dnaigle,ra, nadlsoa nktni-oiwnflna amsm haetnobraynee,f fheactss b[e1e4n].
Hutyiloiszceydam inu sCnhiigneer,sael stroakdnitoiownnala sthheernabpayn aes, hwaeslbl eaesn inu tTiliibzeetdanin mCehdiniceisnee t[r1a5d]i.t iHon. naligtehre hraapsy baesenw uelsleads ains
Ta ibseedtaantimvee daincdin esl[e1e5p]. aHg.ennitg e[r16h]a. sHbeyeonscuysaemdiansea (s4e) daantidv escaonpdosllaemepinaeg e(3n)t [a1r6e] .thHey odsocmyainmainnte T(4A)sa nodf
Hsc.o npioglearm ainnde (b3o)tahr emtheteadboomlitiensa cnatnT Acrsoosfs Hth.en ibgleoroadn-dbrbaoitnh bmaerrtaiebro tliot eesffceacnt ctrhoes scethnetrballo noedr-vborauisn sbyasrtreimer
[t1o7e]f.f Seccotpthoelacmenitnrea l(3n)e hrvaos umsosryes tpeomte[n1t7 p].hSacrompaoclaemutiincael( a3c)thivaistym worheepno ctoenmtppahraerdm taoc heyuotisccaylaamctiinveit y(4w) ahnedn
ecxohmibpiatsr erdeltaotihvyeolys cfyeawmerin seid(4e) eafnfedctesx, hhiboiwtserveelra ttihvee lsycfoepwoelarmsiidnee e(f3f)e cctosn,theonwt eovf esrotlhaneascceoopuosl apmlainntes (3is)
ucosnutaelnlyt omfuscohla lnoawceeor uthsapnla tnhtes hisyuossucyalalmyminue c(h4)l ocownetrentht a[n11t]h. eBhecyaouscsye aomf tinheis(, 4t)hceoren tiesn atn[ 1o1n]g.oBiencga uesffeoortf
ttoh isfu,tllhye ruenidsearnstoanngdo tinhge ebfifoosrytntothfeuslilsy oufn sdceorpsotalanmditnhee (b3i)o saynndt hoetshiesro TfAscso pwoiltahmini ntehe(3 S)oalnadnaoctehaeer (TsAees
wSeicthtiionnt 4h)e. Solanaceae(seeSection4).
TThhee nnaarrccoottiicc pprrooppeerrttiieess ooff ccooccaaiinnee ((11)),, aa TTAA ffrroomm tthhee nnoonn--ssoollaannaacceeoouuss ggeennuuss EErryytthhrrooxxyylluumm,, ccaann bbee
aattttrriibbuutteedd ttoo uunniiqquuee mmooddiiffiiccaattiioonnss ooff tthhee TTAA ccoorree ssccaaffffoolldd tthhaatt aarree nnoott pprreesseenntt iinn TTAAss ffrroomm ssoollaannaacceeoouuss
ppllaannttss.. TThhee ccaarrbbooxxyylliicc aacciidd mmeetthhyyll eesstteerr pprreesseenntt aatt tthhee CC22 ppoossiittiioonn iiss rreessppoonnssiibbllee ffoorr tthhee bbiinnddiinngg ooff
ccooccaaiinnee ((11)) ttoo tthhee ddooppaammiinnee ttrraannssppoorrtteerr [[1188]].. CCooccaaiinnee ((11)) hhaass aallssoo bbeeeenn rreeppoorrtteedd ttoo bblloocckk tthhee rreeuuppttaakkee
ooff nnoorr--eeppiinneepphhrriinnee,, sseerroottoonniinn ((55--HHTT rreecceeppttoorr)) aanndd ddooppaammiinnee ((DD--AA rreecceeppttoorr)) bbyy tthhee bbiinnddiinngg ooff tthhee
aarroommaattiicc rriinngg pprreesseenntt aatt tthhee 33ββ ppoossiittiioonn ooff tthhee mmoolleeccuullee ttoo ssppeecciiffiicc ssiitteess iinn tthheessee rreecceeppttoorrss,, aaffffeeccttiinngg tthhee
nnoorrmmaall pphhyyssiioollooggyy ooff tthhee cceennttrraall nneerrvvoouuss ssyysstteemm [[1199]].. TThhiiss sstteerreeoossppeecciiffiicc ccoonnffoorrmmaattiioonn iiss ddoommiinnaanntt iinn
TTAAss ffoouunndd iinn tthhee EErryytthhrrooxxyyllaacceeaaee,, bbuutt iiss oonnllyy aa mmiinnoorr ccoonnssttiittuueenntt iinn ssoollaannaacceeoouuss ppllaannttss..
While no direct studies regarding the anticholinergic effects of GAs have been reported, a
computational and NMR based study comparing the structures of TAs and GAs revealed that GAs
Molecules2016,21,1510 3of25
While no direct studies regarding the anticholinergic effects of GAs have been reported,
a computational and NMR based study comparing the structures of TAs and GAs revealed that
GAsadoptanN-axialformthatissimilartomanyknownTAs[20]. TheN-methylgroupintheaxial
conformationisthoughttobethepharmacophoreforTAs[21].Additionally,thegranataneringsystem
providesthesemisyntheticintermediateforthepotentantiemeticagentsDolasteronandGranisetron,
which are serotonin 5-HT receptor agonists [22,23]. These compounds are used as medicines to
3
preventacutenauseainpatientsundergoingchemotherapyandradiotherapyforthetreatmentof
cancer[24].
GAsandTAshavedifferentphysiologicaleffectsbeyondthecentralnervoussystem. UnlikeTAs,
GAsexhibitanti-proliferativeeffectsonhepatomacells[24]. Specifically,murinehepatoma(BNLCL.2)
andhumanhepatoma(HepG2)celllineswereculturedwithvariousdosesofcrudeGA-containing
alkaloidfractionextractsfromSedumsarmentosum. Inhibitionofexcessivegrowthoftumorcellswas
observed,indicatingthatthesecompoundspossessanti-cancerproperties[25]. Otherphysiological
effectsthatdistinguishGAsfromTAsincludetheuseofGAsasananti-wormtreatment. Sincethe
isolationofthefirstGAs,thesecompoundshavebeenclaimedtopossessanti-worm(anthelminthic)
capabilities, which were then studied in detail by scientists in the University of Amsterdam in
1956. These studies focused on deriving which GAs possessed the highest anthelminthic activity.
Theanthelminthicactivityofsyntheticgranataneanditsderivativesweremeasuredinliverfluke.
Their results rendered the highest anthelminthic activity to the compound isopelletierine [26].
Thesefindingswerelaterscientificallysupportedin1963withnewerandbetterchemicalmethods[27].
Fascioliasis,adiseasecausedbyliverflukeFasciolahepatica,iscommonincattle. Molluscidalactivityin
pomegranatebarkextractswaseffectiveinkillingofLymnaeaacuminata,thevectorforF.hepatica[28,29].
Beyondtheirmedicinalproperties,GAsalsodifferfromTAsinthatGAshavebeenfoundtobeuseful
inthepreventionofcorrosionintheoil,gasandmetalindustries[30].
1.2. TheScatteredDistributionofTropanesandGranatanesamongstAngiosperms
TropanealkaloidsarecommonlyfoundinthegenusErythroxylumoftheErythroxylaceaefamily.
The Erythroxylum genus includes at least 230 species that are distributed throughout the tropical
regionsofSouthandCentralAmerica[3,31,32]. Erythroxylumcocawasoneofthefirstdomesticated
plantspeciesthatprovidednutritional,medicinal,anddigestivepropertiestoancientcivilizations
bychewingtheleavesoftheplant[33]. Mostofthecultivatedcocausedforcocaine(1)production
comesfromthisspecies[34]. AlbertNeimannfirstisolatedcocaine(1)asapuresubstancein1860[35]
and its use exploded in popularity following an endorsement by Sigmund Freud [36]. The leaves
ofErythroxylumnovogranatensewerealsochewedbytheeliteclassfortheirhighcontentofmethyl
salicylate, which imparts a minty taste [37,38]. This species is known as “Colombian coca” and is
foundtobecultivatedinthemountainsofpresentdayColombia. “Trujillococa”(E.novogranatense
var. truxillense)isacultivarthatisgrownindryandaridregions. Thisspeciesisalsorichinmethyl
salicylate and contains other flavoring qualities that are still used in the production of Coca Cola,
howevertodaytheextractsaredecocainized[34].
Atropine,scopolamine(3),andhyoscyamine(4)areafewwell-knownTAsfromtheSolanaceae
family. Asdiscussedabove,thesecompoundsarecommonlyfoundinspeciessuchasAtropabelladonna,
H. niger, and many members of the genus Datura. Scopolamine (3) was first isolated in 1888
from Scopolia japonica [39]. In medieval Europe, extracts from A. belladonna, were used as poisons,
hallucinogensandaphrodisiacs[17]. FivetotenberriesofA.belladonnacouldkillaperson. Thetoxicity
oftheextractshasalsobeenusedonarrowstopoisonvictims[40]. WhenextractsofA.belladonnaare
appliedtotheeyes,dilationofthepupilsoccurs[41]. Forthisreason,womenusedA.belladonnaas
acosmeticdrugduringtheRenaissance. Womenofthe15thcenturywhoweredevotedtowitchcraft
alsoexploitedthepsychoactiveeffectsofA.belladonna[16]. Mucousmembranes,suchasthosefound
inthewallsoftheoralcavityandthevulva,arereadilysusceptibletodrugabsorption. Itisbelieved
that the application of alkaloid-containing salves to the skin or vulva was achieved by the use of
Molecules2016,21,1510 4of25
brooms. It gave users the feeling of being able to fly, feeding the folkloric associations of witches
withbrooms[17]. Atropinewasfirstisolatedin1833fromA.belladonna[42,43]. Thecorrectstructure
ofatropinewasobtainedbyWillstätterin1889aftermuchdeliberationandstructuralstudies[17].
LeavesofD.metelfromsolanaceousplantswereusedasherbalcigarettesinthe19thand20thcenturies
totreatpatientswithasthmaorotherrespiratoryconditions[17].
GAsincludepelletierine(5),isopelletierine,pseudopelletierine(6),andN-methylpelletierine(7)
and their dMoelerciuvlesa 2t0i1v6,e 2s1, a15n10a basine (8) and anaferine (9) (Figure 2). GAs are predomina4n otf l2y4 found in
P. granatum although they have been characterized in other species such as S. sarmentosum, and
GAs include pelletierine (5), isopelletierine, pseudopelletierine (6), and N-methylpelletierine (7) and
Withania somnifera [44–47]. The pomegranate tree is native to Iran, Afghanistan, Baluchistan, and
their derivatives anabasine (8) and anaferine (9) (Figure 2). GAs are predominantly found in P. granatum
HimalayasaltihnouNgoh rththeye rhnavIen bdeeian .chPaoramcteergizreadn ian toethcear nspaeclsieos sbuechf oasu Sn. sdarimnentthoseumM, eanddi tWeritrhaanniea asonmaninfedra Caucasus
regionsdu[e44t–o47i]t.s Tahne pcioemnetgcraunlatitev tarteieo ins n.aTtoivde atoy ,Irpano,m Afegghraannisatatne, iBsalcuuchltiisvtaant,e adnda HlliomvaelaryaIns idni Na,oSrtohuertnh eastAsia,
India. Pomegranate can also be found in the Mediterranean and Caucasus regions due to its ancient
Malaysia,theEastIndies,tropicalAfrica,andtheUnitedStates[48]. In1879,FrenchchemistsTanret
cultivation. Today, pomegranate is cultivated all over India, Southeast Asia, Malaysia, the East Indies,
andPelletierisolatedabasicsubstancefromtherootbarkofthepomegranatetreeandcharacterized
tropical Africa, and the United States [48]. In 1879, French chemists Tanret and Pelletier isolated a basic
thesalt[45su].bsAtasncoe ffrtohmis three rvoioet wba,rnk oof sthtue pdoimesegrreagnaatred trineeg ancdh cahraarcatceterriizzeadt itohne soalft [a4n5]y. Asst rouf tchtius rreavliegwe,n esorthe
enzymes rneos pstoudniseisb rleegafrodrintgh cehaprraoctderuizcattiioonn oof faGnyA sstruhcatusrabl egeennesr eopr othrete edn.zyImnetse rreessptoinngsilbyle, tfohre thise olation of
production of GAs has been reported. Interestingly, the isolation of pseudopelletierine (6) has been
pseudopelletierine(6)hasbeenreportedinthespeciesErythroxylumlucidum[49]suggestingthatGAs
reported in the species Erythroxylum lucidum [49] suggesting that GAs and TAs may use similar
andTAsmayusesimilarbiosyntheticmachinery.
biosynthetic machinery.

Figure 2. Examples of granatane alkaloids and granatane alkaloid derivatives found among several
Figure2.Examplesofgranatanealkaloidsandgranatanealkaloidderivativesfoundamongseveral
plant species.
plantspecies.
Scientific reports on the occurrence and distribution of TAs from angiosperm families other than
the Solanaceae and Erythoxylaceae are few. When viewed against a phylogeny of angiosperms, the
ScientificreportsontheoccurrenceanddistributionofTAsfromangiospermfamiliesotherthan
results reveal a scattered and non-contiguous distribution (Figure 3) [50,51]. For example, TAs have
theSolanaceaeandErythoxylaceaearefew. Whenviewedagainstaphylogenyofangiosperms,the
been reported in members of the family Proteaceae. Compounds found in the family Proteaceae include
resultsrevtehae lpayrasncoattrtoepraendesa, sntrdobnamonin-ec aonndt bigeluleonudisned aiss wtreilbl uast tihoen co(mFipgouunreds3 f)er[r5u0g,i5ni1n]e. aFnodr feeruxgaimnep [5l2e],. TAshave
been repoIrnt eadddiintiomn, ae mfewb emresmobfertsh weitfhainm thiley faPmriolitees aBcraesaseic.acCeaoe manpdo Cuonnvdoslvfuolaucneade hinaveth beeenfa fmouinldy toP roteaceae
produce calystegines, which are heavily hydroxylated forms of TAs [53]. The main TA producing
includethepyranotropanes,strobamineandbellendineaswellasthecompoundsferruginineand
species are scattered among four orders, which include the Solanales, Malpighiales, Proteales and
ferugine[52]. Inaddition,afewmemberswithinthefamiliesBrassicaceaeandConvolvulaceaehave
Brassicales. In the case of GAs, P. granatum from the family Lythraceae is the main producing species.
beenfoundHotwoepvreor,d thuec aepcpaealyrasntceeg oifn GeAs,s whahs iaclhso abreeenh reeapvoritleyd hiny mdermoxbyerlsa otef dthef oSormlansacoefaeT,A Crsa[s5su3l]a.ceTahe,e mainTA
producingasnpde Ecriyetsharorxeyslaccaetatee rfaemdialimes.o Tnhgesfeo fuamroilriedse bresl,owngh tioc thhein ocrdluerdse MthyretaSleosl,a Snoalalneasl,eMs, Malaplpigighhiiaalleess, ,Proteales
and Saxifragales. This distribution pattern raises the important question about the biosynthetic origin of
andBrassicales. InthecaseofGAs,P.granatumfromthefamilyLythraceaeisthemainproducing
the respective GA and TA pathway. Namely, have these biosynthetic pathways arisen from a common
species. However, the appearance of GAs has also been reported in members of the Solanaceae,
ancestor or have they arisen independently in several cases? Recent evidence in TA biosynthesis
Crassulacesaugeg,easntsd thEatr ycetrhtarionx byiolasycnetaheetifca smteiplsi ehsa.vTe hmeuslteipflae moriigliienss [b5e].l Boyn egxtteonstihoen, othrids ecrosulMd aylsrot amleeasn, Solanales,
Malpighiatlheast, GaAn bdioSsyanxtihfersaisg haalse sar.isTenh iinsddepiesntrdiebnutltyi omnorpe tahtatne ronncrea. i ses the important question about the
biosyntheticoriginoftherespectiveGAandTApathway. Namely,havethesebiosyntheticpathways
arisenfromacommonancestororhavetheyarisenindependentlyinseveralcases? Recentevidencein
Molecules2016,21,1510 5of25
TAbiosynthesissuggeststhatcertainbiosyntheticstepshavemultipleorigins[5]. Byextension,this
couldalsomeanthatGAbiosynthesishasarisenindependentlymorethanonce.
Molecules 2016, 21, 1510 5 of 24

FigureFi3g.uTreh 3e. dTihvee drisviteyrsaitny dansdim siimlairlaitriietiseso offt trrooppaannee aanndd ggrarnaantaatnaen peropdroudciungci annggiaonsgpeiormspse. rTmhes .orTdheerso rders
tropane alkaloids belong to are highlighted in orange. The orders granatane alkaloids belong to are
tropanealkaloidsbelongtoarehighlightedinorange.Theordersgranatanealkaloidsbelongtoare
highlighted in blue. Shared orders are represented by both colors. The plant families that are known
highlightedinblue.Sharedordersarerepresentedbybothcolors.Theplantfamiliesthatareknownto
to produce tropane and granatane alkaloids are branched off from the orders. The scale at the bottom
producetropaneandgranatanealkaloidsarebranchedofffromtheorders. Thescaleatthebottom
represents millions of years. Modification of the phylogenetic tree published in [54], republished with
represpeenrtmsimssiiollnio wnistho fthyee Baortsa.nMicaold Sioficcieattyio onf Aofmtehreicpa.h ylogenetictreepublishedin[54],republishedwith
permissionwiththeBotanicalSocietyofAmerica.
Examples of scattered distributions of alkaloid classes can be observed throughout the plant
kingdom. For example, the legumes (Fabaceae) contain several alkaloid secondary metabolites that are
Examples of scattered distributions of alkaloid classes can be observed throughout the plant
not found in all members within the family [55]. In some cases, de novo evolution of the pathways have
kingdom. Forexample,thelegumes(Fabaceae)containseveralalkaloidsecondarymetabolitesthatare
been observed. In the case of indolizidine alkaloids, the source of the scattered appearance of alkaloids
notfoundinallmemberswithinthefamily[55].Insomecases,denovoevolutionofthepathwayshave
is theorized to occur due to horizontal gene transfer from alkaloid producing endophytic fungi.
beenobserved. Inthecaseofindolizidinealkaloids,thesourceofthescatteredappearanceofalkaloids
istheo1r.3iz. eBdiostyonothcecsuis rofd TuAest aondh oGrAizs ontalgenetransferfromalkaloidproducingendophyticfungi.
Diversity among tropane and granatane alkaloids can also be seen at the biochemical level. Most data
1.3. BiosynthesisofTAsandGAs
available regarding enzymes involved in TA biosynthesis comes from species within the Solanaceae
Dfaivmeirlys.i tDyeaspmitoen rgecternot paadnveanacnemdegnrtsa,n saotmane ecraitlikcaall ositdepssc iann TaAls obiobseynsteheensisa rtetmhaeinb iaomchbiegmuoiucas.l level.
Most data available regarding enzymes involved in TA biosynthesis comes from species within
Molecules2016,21,1510 6of25
theSolanaceaefamily. Despiterecentadvancements,somecriticalstepsinTAbiosynthesisremain
ambiguous.Additionally,recentadvancesinhighthroughputsequencingtechnologies,plantgenomics,
Molecules 2016, 21, 1510 6 of 24
andbiochemicalmethodshaveaidedtheprogressofelucidatingTAbiosynthesisinothernon-model
specieAsd,dsitpioencaifillyc,a lrleycewnti thadinvatnhcees Eirny thhigrohx ythlraocuegahep.utL asesqtluye,necvinegn ttehchonuoglohgitehs,e pbliaonst ygnetnhoemsiiscso, fanGdA s is
predicbtioecdhteombiceasl immeitlhaordtso hTaAves ,apidleadu sthibe lpyroagwrehsso olef enleuwcidsaettinogf TeAnz byiomsyenstwheesrise irne ocrthueirt endonfo-mrtohdeelb sipoescyienst, hesis
of GAspse.cTifihcaisllyw woiuthldin mtheea Enrytthharotxuynlatcaepaep. eLdasgtleyn, eevaenn dtheonuzgyh mthee dbiiovseyrnstihteysiiss opf rGeAsesn its ipnretdhiceteGdA to abned TA
similar to TAs, plausibly a whole new set of enzymes were recruited for the biosynthesis of GAs. This
biosyntheticpathways. Itisthereforeimportanttoelucidatethebiosynthesisofthesealkaloidstohelp
would mean that untapped gene and enzyme diversity is present in the GA and TA biosynthetic
furtherunderstandspecificpartsoftheirmechanismsandtheirmetabolicprocesses.
pathways. It is therefore important to elucidate the biosynthesis of these alkaloids to help further
understand specific parts of their mechanisms and their metabolic processes.
2. TropaneAlkaloidBiosynthesis
O2.v Terroaplla,nthe eAplkroalcoeisds Boifopsyrondthuecsiinsg eitherTAsorGAsinplantsbeginswiththediversionofspecific
amino acids from primary metabolism into the formation of an initial nitrogen-containing ringed
Overall, the process of producing either TAs or GAs in plants begins with the diversion of specific
compaomuinndo .aTcihdiss fhroemte rporciymcalreyw mileltainbomlisoms ticnatos etshep rfoocrmeeadtioonn otfo afno rimnitaialb inciytrcolgicens-tcrouncttauinriengin rwingheicdh the
coresckoemleptoounnsda. rTehicsr heaetteerdoc(yFcilgeu wriell 1in). mFousrtt chaesresm porodciefiecda otino tnos fowrmill aa bdidcydcliivc esrtrsuecftuurnec itnio wnhailchg rtoheu cposreto the
coresstkreulecttounrse ayreie clrdeiantegdt (hFeigfiunrea 1l)e. nFudrtphreord muocdt.ifiOcartnioitnhs iwneill( a1d0d) adnivderasreg fiunnicnteio(n1a1l )graorueptsh teo athme icnoorea cids
prediscttreudctutore byeieltdhiengs ttahret fiinngal seundb sptrroadteuscto. OfrTnAithbinioe s(y10n)t ahneds iasrg(iSncihnee m(11e) 1ar)e[ t5h6e] .amFieneod aicnidgs sptrueddiicetsedu sing
14C-ptroo blien tehien sttoartthinegr osuobtsstoraftAes. obfe lTlaAd obnionsayanltshoessius g(Sgcehsetmthea 1t) p[5r6o]l.i Fneee(d1i2n)g mstuaydibese uassintagr 1t4iCn-gpraomliinneo inatcoi dfor
the roots of A. belladonna also suggest that proline (12) may be a starting amino acid for incorporation into
incorporationintothetropanemoiety. OtherstudiesusingD.metelandD.stramoniumhavealsoshown
the tropane moiety. Other studies using D. metel and D. stramonium have also shown the incorporation of
theincorporationofproline(12)intoTAcompounds,suchasscopolamine(3)andtropine(13)[57].
proline (12) into TA compounds, such as scopolamine (3) and tropine (13) [57]. Biosynthetically each
Biosyntheticallyeachofthesethreeaminoacidscangenerateapyrroline-5-carboxylateintermediate,
of these three amino acids can generate a pyrroline-5-carboxylate intermediate, which can be
whichcanbeinterconvertiblebetweentheseaminoacids[58]. Consequently,feedingstudiesoflabeled
interconvertible between these amino acids [58]. Consequently, feeding studies of labeled amino acids
aminoarae cdidiffsicaurlet tdoi fifintceurlptrteot winittheropurt eftuwrthitehr oeunztyfumrotlhoegriceanl zdyamta.o logicaldata.

SchemScehe1m.Ter 1o.p Tarnoepaanlke aallokiadlobidi obsiyosnytnhtehseissisu uppt otot hthee ffoorrmmaattiioonn oof fththe eNN-m-methetyhl-yΔl1--∆py1-rproylirnrioulmin icuamtionc.a tion.
Arginine, ornithine and proline are interconvertable, sharing the pyrroline-5-carboxylate intermediate
Arginine,ornithineandprolineareinterconvertable,sharingthepyrroline-5-carboxylateintermediate
(highlighted in blue). Putrescine can be formed directly via decarboxylation of ornithine or indirectly
(highlightedinblue).Putrescinecanbeformeddirectlyviadecarboxylationofornithineorindirectly
through arginine. Methylation of putrescine is followed by oxidation to yield 4-methylamino-butanal,
througharginine.Methylationofputrescineisfollowedbyoxidationtoyield4-methylamino-butanal,
which is then spontaneously cyclized to yield the N-methyl-Δ1-pyrrolinium cation. Enzymes are
whichhiigshltihgehntedsp ino onrtaanngeeo aunsdly abcbyrcelviizaetedd taos fyoileloldwst:h AeDNC-,m aregtihniynle- ∆d1e-cparybrorxoylilansieu; mAIHca, taigomn.atiEnne zimymineos are
highlihgyhdtreodlaisne;o NraCnPgAeHa,n Nd-acabrbbraemvoiaytlepdutarsesfcoilnleo wams:inAoD hCyd,raorlgaisnei;n OeDdCe,c aorrbniothxyinlea sdee;cAarIbHo,xayglamsea; tPinMeTi,m ino
hydropluatsree;scNinCe PNA-mHe,tNhy-lc tarrabnasfmeroaysel;p MutPrOes, cmineethayml piuntorehscyidnero olxaisdea;sOe. DC,ornithinedecarboxylase;PMT,
putrescineN-methyltransferase;MPO,methylputrescineoxidase.
Molecules2016,21,1510 7of25
Anonsymmetricalintermediatehasbeenproposedfortheproductionofthepyrrolidinering,
iftheaminoacidornithine(10)isfirstmethylatedattheγ-Nposition. Aproposedalternativeroute
includes the decarboxylation of ornithine (10) to form the polyamine putrescine (14) as the first
step[59,60]. Feedingstudiesofradioactivelylabeledornithine-2-14ChaveshownthatseveralDatura
speciesincorporateanonsymmetricalintermediate[59]. However,asymmetricalintermediatehas
beenreportedforNicotiana,Erythroxylum,andHyoscyamusspecies[61]. Symmetricalincorporation
showedactivityatpositionsC1andC5ofthetropaneringandhasbeenreportedforNicotiana,E.coca,
and Hyoscyamus albus [59,60]. A one-step enzymatic approach is possible to reach a symmetrical
intermediatebyconvertingornithine(10)intoputrescine(14)catalyzedbyornithinedecarboxylase
(ODC)[62]. Anotherroutetoputrescine(14)canbetakenbystartingwiththeaminoacidarginine(11).
Thedecarboxylationofarginine(11)intoagmatine(15)iscatalyzedbyargininedecarboxylase(ADC).
Agmatine(15)canthenbeconvertedintoN-carbamoylputrescine(16)viaagmatineiminohydrolase
(AIH).TheenzymeN-carbamoylputrescineamidohydrolase(NCPAH)thenyieldsputrescine(14).
BoththeADCandODCdirectedpathwayshavedifferentanddiverseoutcomesinregardstoprimary
andsecondarymetabolism. Putrescine(14)thatwasproducedbyODCisimportantforthesupply
of polyamines for primary metabolic processes such as cellular differentiation, development and
division [63]. On the other hand, putrescine (14) that was produced by ADC in the Solanaceae is
thoughttoberequiredforenvironmentalstressrelatedresponses.
TheformationofN-methylputrescine(17)catalyzedbyputrescineN-methyltransferase(PMT)
is considered the first rate-limiting step in the TA biosynthetic pathway [64]. PMT is an
S-adenosylmethionine (SAM)-dependent methyltransferase that attaches a methyl group to the
nitrogenatomthatultimatelyappearsinthetropaneskeleton. ThefirstPMTsequencetobeisolated
fromplantscamefromtobacco(Nicotianatabacum)[65]. Sincethen,manyotherPMTrelatedsequences
havebeenisolatedandcharacterizedfromotherpyrrolidinealkaloidproducingplantspecies. PMT
belongs to a large family of enzymes that are involved in polyamine production. These primary
metabolites play an important role in stress physiology, senescence, and morphogenesis [66].
Several lines of evidence suggest that PMT function has evolved from an ancestral spermidine or
sperminesynthases[67]. Thiswouldsuggestthatanenzymefromprimarymetabolismwasrecruited
for the production of secondary metabolites. Localization of the PMT protein is root specific for
both nicotine and tropane producing solanaceous plants [68–70]. While N-methylputrescine (17)
is the predominant intermediate for solanaceous TAs, at least one alternative has been suggested.
[6-14C]-1,5,10-triazadecanefedtoNicotianaglutinosaplantsrevealedthatN-methylspermidinecanalso
beincorporatedintothepyrrolidineringofnicotine[71].
The next step in TA biosynthesis is the formation of 4-methylamino-butanal (18) from
N-methylputrescine(17)catalyzedbymethylputrescineoxidase(MPO).Thisenzymecoexistswith
aclassofcopper-dependentdiamineoxidasesthatusescopperasacofactortooxidizeaconserved
tyrosineresidueintoatopaquinone,essentialforenzymecatalysis[72]. MPOwasfirstcharacterized
fromthespeciesN.tabacum[73,74]. AlthoughanMPOlikegenehasyettobediscoveredinE.coca,
thecorrespondingactivityfromintactplantsfed4-monodeuteratedN-methylputrescineshowsthe
same enantioselectivity as solanaceous plants that produce either nicotine or TAs [75]. Using 13C
fractionationtechniques,researchershavefoundthatbothnicotineandhyoscyamine(4)sharethe
samebiosyntheticpathway,atleastuptotheN-methyl-∆1-pyrroliniumcation(19)[76]. Subsequently,
ahypothesishasbeenproposedthatametabolicchannelprevailsinwhichamulti-enzymecomplex
is active. The product of MPO, 4-methylamino-butanal (18), spontaneously cyclizes to form the
N-methyl-∆1-pyrrolinium cation (19) [59]. Feeding studies using ornithine-2-14C detected labeled
4-methylamino-butanalinD.stramoniumplants[59]. TheN-methyl-∆1-pyrroliniumcation(19)serves
asthefirstringinthebicyclictropaneskeleton.
WhilethebiochemicalroleofPMTsandMPOsaregenerallyacceptedstepsleadingtothefirst
ringclosureoftropaneintermediatesintheSolanaceae,theenzymaticbasisofthesecondringclosure
iscontroversial. Formanyyears,thecompoundhygrine(20),(R)-1-(1-methylpyrrolidin-2-yl)-propan-
Molecules2016,21,1510 8of25
2M-oolneceulwes a20s1t6h, 2o1u, g15h1t0 tobeadirectintermediatebasedonfeedingstudies,howevermorerecentstu8 dofi e2s4
have demonstrated that previous results are likely experimental artifacts [77–79]. In solanaceous
ppllaannttss tthhee bebsets itncionrcpoorrpaotiroanti oinntoi nthteo stehcoensde rcionngd wraisn agchwieavseda cfrhoimev eradcefmroimc etrhaycle [m2,3ic-13eCt2h]-y4l-([N2-,3m-1e3tChy2l]--
42--(pNy-rmroelitdhiynly-l2)--p3-yorxroobliudtiannyola)-t3e- [o7x7o,8b0u],t aan pooalyteke[ti7d7e,8 b0a]s,eda mpoolelycukleet i(dSechebmasee 2d). Fmuortlheecru elevid(Senchcee mfoer th2)e.
Finuvrothlveermeevnidt eonfc ea pfoorlytkheetidine vwolavse mdeemntonosftraatepdo lbyyk etthidee fewedaisngd eomf omnesttrhaytle d(RSb)y-[1t,h2e-13fCe2e,1d-i1n4Cg]-o4f-
m(1e-mtheytlh(yRl-S2)--p[1y,r2r-o13liCdin,1y-1l)4-C3-]o-4x-o(1b-umtaenthoyatle-2 t-op ytrhreo lliedaivneysl )o-3f -oEx. ocbocuat a[n81o]a. tTehtoist hsterolenagvleys iomfpEl.iccaotceas[ 8th1]e.
2
Tinhvisolsvteromnegnlyt oimf apcleictaattee-sdtehreiviendv omlveetambeonlitteosf (aec.get.,a pteo-ldyekreivtieddesm) detuarbinogli ttehse( efo.grm.,paotiloynk eotfi dthees )sdecuornindg rtihnge
fionr TmAasti.o Tnhoefret haeres elicmonitdedri tnygpiens ToAf es.nTzyhmereesa craepliambliete odf tcyaptaelsyozfinegn zthyims etyspcea poaf bcloenodfecnastaatliyoznin rgeatchtiisotny. pIne
othfec ocnadsee nosfa tbioennzreyalicstoioqnu.inInoltihnee caalskeaolofibde nbzioysliysnotqhueisnios lian ePiacltkeatl–oSipdebnigolseyrnatshee seinszayPmicet ewt–aSsp sehnogwlenra steo
ecnonzydmenesew dasopshamowinne twoictohn 3d,e4n-dseihdyodproaxmyipnheewnyitlhac3e,t4a-lddiehhyyddreo x[y82p]h. eHnoywlaecevtearl,d beahsyedde o[n82 m].oHreo wreecveenrt,
bisaosteodpe oannd mraodrieolarbeecelendt feiseodtionpge stuadnides rita idsi ohlyapboetlhedesizfeeedd tihnagt as ttyupdeie IsII pitolyiskehtiydpe ostyhnetshizaesed (PtKhaSt)
aretaycptieonI IiIs ipnovloylkveetdid ine csoynndthenassein(gP aKcSet)artee aucntiiotsn onistoi nthveo Nlv-emdeitnhyclo-Δn1d-penyrsrinoglinaiucemta ctaetiuonni (t1s9o) nletaodtihneg
Nto- mtheet sheyclo-∆nd1- priynrgr ocllionsiuurme ocfa TtiAon b(i1o9s)ylnetahdeisnisg [t7o7t,8h0e,8s3e]c.o Tnydprei nIIgI PclKosSus raereo faT fAambiiloys oyfn ethnezsyims[e7s7 k,8n0o,8w3n].
Ttoy pcaetIaIlIyPzKe tShsea irteeraatfiavme idlyecoafrebnozxyymlateisvke ncoonwdnentosactaitoanly ozfe mthaelointeyrla-CtiovAe doenctaor CbooxAy-ltaettihveerceodn sduebnsstraatitoesn.
oTfympea IlIoIn PyKl-SCso aAre opnrotomCisocAuo-tuest heenrzeydmseusb tshtarta hteasv.eT ay bpreoIaIdI PtoKleSrsanarcee fporro dmivisecruseo ususbesntrzaytmese asntdh aarteh aabvlee
atob craotaadlytzoel emraunltcipelfeo rredacivtieornsse [s8u4b,8s5t]r.a Tteyspaen IdII aPrKeSasb tlheatto ucsaet caylyclziec nmiturlotgipelne-croenactatiionninsg[ 8su4,b8s5t]r.aTteysp heaIvIeI
PbKeeSns pthraetvuiosuesclyy cclihcanraitcrtoegriezne-dc ofnotra itnhienirg rsoulbess trina teaslkhaaloviedb pereondpurcetvioionu [s8ly6–c8h8a].r aHctoewriezvederf, ourntlhikeier trhoelesse
ipnreavlkioaulosi dstpurdoidesu cthtieo npr[8ed6–ic8t8e]d. Hsuobwstervaetre, iunn TlikAe mtheetsaebpolriesvmio, uNs-smtuedthieysl-tΔh1e-ppyrrerdoilcitneidumsu bcasttiroante (i1n9)T Ais
mcheatragbeodli samnd, Nla-cmkse tah yClo-∆A1 t-hpiyorersotleinr.i umcation(19)ischargedandlacksaCoAthioester.

SScchheemmee 22.. TTrrooppiinnoonnee ffoorrmmaattiioonn ffrroomm tthhee NN--mmeetthhyyll--∆Δ11--ppyyrrrroolliinniiuumm ccaattiioonn iinn wwhhiicchh tthheerree aarree ttwwoo
ppoossssiibbiilliittiieess ffoorr ththee ccoonnddenensastaiotino nofo tfhteh etrotrpoapnaen reinrgin. gA.ceAtycle-CtyolA-C iosA utiislizuetdil izveiad avcieatoaacceettoataec etota tyeietlod
yhiyegldrinhey-g1r-cinareb-1o-xcyalricb oaxciydl.i cOanc itdh.eO onthtehr ehoatnhde,r twhaon sdu,ctcwesosisvuec dceescsairvbeoxdyelcaatrivbeo xcoynladteivnesactoionndse onfs amtiaolnonsyolf-
mCoaAlo nbyyl -PCKoSA (pboylyPkKeStid(peo slyyknethtiadsee)s yynietlhdass 4e)-(y1-iemldetsh4y-l(-12--mpyertrhoylli-d2i-npyylr)-r3o-loidxionbyult)a-3n-ooyxlo-CbuotAa.n Eoynlz-yCmoAes.
Earnez yhmigehsliagrhetehdig ihnl iogrhatnegdei nanodr asnugbestarnatdess uabres thraigtehsliagrhetehdig ihnl ibglhutee.d inblue.
SSppoonnttaanneeoouuss ddeeccaarrbbooxxyyllaattiioonn aatt tthhee CC22 ppoossiittiioonn ffoolllloowwiinngg rriinngg cclloossuurree ccaann bbee pprreevveenntteedd bbyy tthhee
ffoorrmmaattiioonn ooff tthhee mmeetthhyyll eesstteerr.. TThhiiss wwoouulldd eexxppllaaiinn tthhee pprreesseennccee ooff tthhee ccaarrbboommeetthhooxxyy ggrroouupp ffoouunndd iinn
ccooccaaiinnee ((11)) aass wweelll laass ooththere rtrtorpoapnaen ealaklakloaildosid psropdroudceudc ebdy bpylapnltas nftosufnodu nind thine tEhreytEhrryotxhyrloacxeyalea.c eTahee.
Trehseulrteinsgu lctoinmgpocoumndp mouenthdylmecegtohnyolneceg (2o1n)o, cnoent(a2i1n)s, ac koenttoa ifnusncatiokne taot tfhuen Cc3ti poonsiatitotnh aendC 3a cpaorbsiotmioenthaonxdy
agrcoaurbpo amt eththeo Cxy2 gproosuitpioant [t5h]e. RCe2dpuocstiitoionn a[t5 C].3R ies dnueccteisosnarayt Cfo3r iessnteerc efossramryatfioorne tsot eorcfcourrm. ationtooccur.
FFoorr tthhee mmeemmbbeerrss ooff tthhee SSoollaannaacceeaaee,, ttrrooppiinnoonnee rreedduuccttaassee ((TTRR)) eennzzyymmeess ccaattaallyyzzee tthhee rreedduuccttiioonn ooff
kkeettoo ggrroouuppss iinn tthhee ttrrooppaannee rriinngg.. TThheessee eennzzyymmeess aarree ppaarrtt ooff tthhee sshhoorrtt cchhaaiinn ddeehhyyddrrooggeennaassee//rreedduuccttaassee
((SSDDRR)) ffaammiillyy tthhaatt cacatatalylyzezse sNNAADD(P()P(H)(H)-d)-edpeepnednednetn mt omnoonmoemriecr iocxiodxoirdeodruedctuacstea sreeacretiaocntsio [n8s9][. 8I9t]s.
Iatcstiavcittiyv ictoynctroonltsr tohlse mtheetambeotlaicb folluicx fltouwxatrodws aTrAd sbiToAsybnitohseysnist hdeoswisndstorweanmst r[9e0a]m. In[9 s0o]l.anInacseooluans apcleaonutss,
ptwlaon ttsy,ptews ooft ytproepsionfontreo preidnuoncteasreesd euxcitsats, etrsoepxiinsot,nter orpedinuocntaesere Id (uTcRt aIs) eaIn(dT tRroIp)iannodnet rroepdiuncotnaseer eIId (uTcRta IsIe).
ITIh(eTyR sIhIa).reT ah ecyomshmaoren atecrotimarmy “oRnotsesrmtiaarny” “foRlods sstmruacntu”rfeo, lad csotnrusecrtvuerde, maoctoifn tsheartv ceodnmsisottsi foft htwatoc poanisriss otsf
α-helices and six parallel β-sheets, a catalytic active site with the motif YxxxK, and a dinucleotide
cofactor-binding motif [91]. These enzymes share more than 50% of amino acid sequence similarity and
are also assumed to have evolved from a common ancestor [90]. As little as five amino acid differences
are needed to change the stereochemistry of the product [92]. TR I only converts the 3-keto function
Molecules2016,21,1510 9of25
oftwopairsofα-helicesandsixparallelβ-sheets,acatalyticactivesitewiththemotifYxxxK,and
adinucleotidecofactor-bindingmotif[91].Theseenzymessharemorethan50%ofaminoacidsequence
sMimoleiclualresit 2y01a6n, d21,a 1r5e10a lsoassumedtohaveevolvedfromacommonancestor[90]. Aslittleasfiveam9 oifn 2o4
acid differences are needed to change the stereochemistry of the product [92]. TR I only converts
tthoe a3 p-kroedtoufcut nthcatito hnatso aa 3pαr-ocodnufcigtuthraattihonas (Sac3hαe-mcoen 3fi).g Tuhraist ipornod(Succheesm treo3p)i.nTe h(1is3)p (r3oαd-utrcoepsatnroopl)i,n weh(1ic3h)
(s3eαrv-terso paas nao pl)r,ewcuhriscohrs feorrv ae swaisdae rparnecguer osfo ersftoerriafiwedid TeAras n[5g]e. Oofne tshteer cifioendtrTaArys, [T5R]. IOI pnrtohdeuccoenst aranr ay,lcToRhoIIl
psoroledluy cwesitahn aa 3lβco-choonlfsigoulerlaytiwonit hcaalle3dβ -pcsoenufidgoutrraotpioinnec (a3lβle-tdroppsaenuodlo).t rTohpiisn ies t(h3βen-t rcoopnavneortle).dT tho idsiifsfetrheennt
cnoonnveesrtteerdifiteodd TifAfesr ecnatllneodn ceasltyesrtifiegeidneTsA. sAc aglelende cdaulypsltiecagtiinoens .eAvegnetn ies dauttprilbicuatteidon toe vtehnetseis tawttor idbiuftfeedretnot
tThResse intw thoed SiofflearneancteTaRe sfainmtihlye [S9o3l]a. naceaefamily[93].

SScchheemmee 33.. TTrrooppiinnoonnee iiss ccoonnvveerrtteedd iinnttoo ttrrooppiinnee vviiaa TTRRII,, ttrrooppiinnoonnee rreedduuccttaassee II.. TTrrooppiinnee uuttiilliizzeess
pphheennyyllllaaccttooyyll--CCooAA ttoo yyiieelldd lliittttoorriinnee,, wwhhiicchh tthheenn uusseess aa ccyyttoocchhrroommee pp445500 eennzzyymmee ttoo ffoorrmm hhyyoossccyyaammiinnee..
SSccooppoollaammiinnee iiss eeppooxxiiddiizzeedd bbyy tthhee eennzzyymmee HH66HH,, 66ββ--hhyyddrrooxxyy hhyyoossccyyaammiinnee eeppooxxiiddaassee,, iinn aa ttwwoo--sstteepp
pprroocceessss.. EEnnzzyymmeess aarree hhiigghhlliigghhtteedd iinn oorraannggee aanndd ssuubbssttrraatteess aarree hhiigghhlliigghhtteeddi innb blluuee..
However in E. coca, a very different type of reductase enzyme was found for the reduction of the
HoweverinE.coca,averydifferenttypeofreductaseenzymewasfoundforthereductionof
3-keto function of methylecgonone (21) (Scheme 4). This is the first evidence of a polyphyletic origin
the3-ketofunctionofmethylecgonone(21)(Scheme4). Thisisthefirstevidenceofapolyphyletic
for TA biosynthesis in plants. Methylecgonone reductase (MecgoR) was purified from crude protein
originforTAbiosynthesisinplants. Methylecgononereductase(MecgoR)waspurifiedfromcrude
extracts from coca leaves using classical biochemical techniques [5]. This enzyme was found to be
proteinextractsfromcocaleavesusingclassicalbiochemicaltechniques[5]. Thisenzymewasfoundto
very different than the TR enzymes that catalyzed the reduction reaction of the 3-keto function in
beverydifferentthantheTRenzymesthatcatalyzedthereductionreactionofthe3-ketofunctionin
solanaceous plants. MecgoR shares an overall identity of less than 10% at the amino acid level when
solanaceous plants. MecgoR shares an overall identity of less than 10% at the amino acid level
compared to any TRs. MecgoR belongs to an aldo-keto reductase (AKR) superfamily of enzymes. AKRs
when compared to any TRs. MecgoR belongs to an aldo-keto reductase (AKR) superfamily of
that have been characterized so far share a common α/β-barrel motif, using either NADH or NADPH
enzymes. AKRsthathavebeencharacterizedsofarshareacommonα/β-barrelmotif,usingeither
as a cofactor [94]. The MecgoR protein is localized in the palisade parenchyma of young developing
NADHorNADPHasacofactor[94]. TheMecgoRproteinislocalizedinthepalisadeparenchymaof
leaves. This is in contrast to the localization of roots in TRs of solanaceous plants. This enzyme is also
youngdevelopingleaves. ThisisincontrasttothelocalizationofrootsinTRsofsolanaceousplants.
similar to other enzymes of alkaloid metabolism such as codeinone reductase and an enzyme of flavonoid
Thisenzymeisalsosimilartootherenzymesofalkaloidmetabolismsuchascodeinonereductaseand
biosynthesis, chalcone reductase. The stereospecific enzyme MecgoR catalyzes the conversion of
anenzymeofflavonoidbiosynthesis,chalconereductase. ThestereospecificenzymeMecgoRcatalyzes
methylecgonone (21) to the 3β-hydroxy-containing compound methylecgonine (22). MecgoR can also
theconversionofmethylecgonone(21)tothe3β-hydroxy-containingcompoundmethylecgonine(22).
use tropinone (23) as a substrate but this only produces pseudotropine, which is consistent in E. coca
MecgoR can also use tropinone (23) as a substrate but this only produces pseudotropine, which
with the presence of only 3β-hydroxy esters. Common TAs are esterified with aromatic or aliphatic
is consistent in E. coca with the presence of only 3β-hydroxy esters. Common TAs are esterified
acids, with the stereochemistry of the hydroxyl group dependent on the reductase used.
witharomaticoraliphaticacids,withthestereochemistryofthehydroxylgroupdependentonthe
reductaseused.
Molecules2016,21,1510 10of25
Molecules 2016, 21, 1510 10 of 24

SScchheemmee 44.. EEccggoonnoonnee--CCooAA eesstteerr uunnddeerrggooeess aa mmuullttii--sstteepp pprroocceessss ttoo yyiieelldd mmeetthhyylleeccggoonnoonnee,, wwhhiicchh iiss
rreedduucceedd bbyy mmeetthhyylleeccggoonnnnee rreedduuccttaassee,,M MeeccggooRRt otop rpordoudcuecem metehtyhlyeclegcognoinnien.eC. oCcoacinaeinies tish etnhefno rfmoremdebdy
bthye tahcey laactiyolnatoiofnm oetfh myleecthgyolneicngeowniinthe bwenitzho yble-CnzooAy.lE-CnozAym. eEsnazryemheigs halirgeh theidghinligohratendg eina nodrasunbgset raanteds
saurebshtirgahtelisg ahrtee dhiignhblilguhet.ed in blue.
The benzoic ester of methylecgonine (22) is cocaine (1). Methylecgonine (22) is a molecule that
Thebenzoicesterofmethylecgonine(22)iscocaine(1). Methylecgonine(22)isamoleculethat
has little physiological activity until it is converted into cocaine (1) [95]. There has been a prediction that
haslittlephysiologicalactivityuntilitisconvertedintococaine(1)[95]. Therehasbeenaprediction
an acyltransferase in E. coca utilizes benzoyl-CoA as the activated acid. This prediction was based on
that an acyltransferase in E. coca utilizes benzoyl-CoA as the activated acid. This prediction was
fbeaesdeidngo sntufedeidesin ugsisntgu dtriaenssu-[s3i-n13gCt,r14aCn]s--c[i3n-n13aCm,1i4cC a]c-icdi nannadm thice aNc-iadceatnydlctyhseteNam-aicneet ythlciyosetsetaerm oifn [e3-t1h3Cio,e14sCte]-r
trans-cinnamic acid [96]. Methylecgonine (22) undergoes esterification with a benzoyl moiety that
of[3-13C,14C]-trans-cinnamicacid[96]. Methylecgonine(22)undergoesesterificationwithabenzoyl
was predicted to utilize benzoyl-CoA as the activated acyl donor [97]. It was not determined if it arises
moiety that was predicted to utilize benzoyl-CoA as the activated acyl donor [97]. It was not
from benzoyl-CoA or benzaldehyde, but the moiety was found to be derived from cinnamic acid [96,97].
determinedifitarisesfrombenzoyl-CoAorbenzaldehyde,butthemoietywasfoundtobederived
Acylation reactions of secondary metabolites in plants are catalyzed by several acyltransferase families,
from cinnamic acid [96,97]. Acylation reactions of secondary metabolites in plants are catalyzed
however only the BAHD acyltransferase is known to utilize the activated acyl-CoA thioesters [98].
by several acyltransferase families, however only the BAHD acyltransferase is known to utilize
TAs are modified through the esterification of the hydroxyl function at the C3 position in the tropane
theactivatedacyl-CoAthioesters[98]. TAsaremodifiedthroughtheesterificationofthehydroxyl
ring. It has been established that in E. coca, the cocaine synthase reaction uses benzoyl-CoA and
function at the C3 position in the tropane ring. It has been established that in E. coca, the cocaine
methylecgonine (22) as substrates. With the CoA-dependent nature of this enzyme along with the
synthasereactionusesbenzoyl-CoAandmethylecgonine(22)assubstrates. WiththeCoA-dependent
reported properties for the tigloyl-CoA:pseudotropine acyltransferase from D. stramonium, it was
nature of this enzyme along with the reported properties for the tigloyl-CoA:pseudotropine
hypothesized that cocaine synthase is a member of the BAHD acyltransferase superfamily [99]. This
superfamily of enzymes is well known to participate in secondary metabolite modification of esters and
amides [98]. Cocaine synthase was found to be capable of producing both cocaine (1) via activated