The sca ﬀ old-forming steps of plant alkaloid biosynthesis

Alkaloids from plants are characterised by structural diversity and bioactivity, and maintain a privileged position in both modern and traditional medicines. In recent years, there have been signi ﬁ cant advances in elucidating the biosynthetic origins of plant alkaloids. In this review, I will describe the progress made in determining the metabolic origins of the so-called true alkaloids, specialised metabolites derived from amino acids containing a nitrogen heterocycle. By identifying key biosynthetic steps that feature in the majority of pathways, I highlight the key roles played by modi ﬁ cations to primary metabolism, iminium reactivity and spontaneous reactions in the molecular and evolutionary origins of these pathways.


Background
Alkaloids from plants have remarkable powers to harm, heal and reveal; this has led to their use in herbal medicine and related practises across continents and cultures for millennia.Alkaloids were foundational in the development of organic chemistry and were among the rst pharmaceuticals developed.They retain a privileged position in modern medicine, used widely to treat pain, cancer, dementia and countless other ailments (Fig. 1). 1,2Despite losing pace to synthetic drug discovery approaches in recent decades, plant alkaloids remain a viable source of bioactive compounds with considerable therapeutic potential. 3t is only through the elucidation of plant alkaloid biosynthesis-determining chemical intermediates, characterising enzymes and sequencing genomes-that we can truly appreciate and understand the synthetic ingenuity of nature in constructing these complex compounds.There has been remarkable progress in the discovery and characterisation of enzymes over the last decade, triggered by advances and expanded use of sequencing technologies. 4,5][8] The investigation of plant alkaloid biosynthesis can lead to new tools and technologies.For example, enzymes can be employed as biocatalysts for the in vitro formation of new-to-nature compounds. 9,102][13] Understanding the genomic basis for alkaloid biosynthesis can also inform efforts to breed plants with higher compound yields or lower toxicity. 14

Denition of alkaloids
Alkaloids were originally dened as alkaline substances extracted from a plant with a biological activity. 15This denition has been rened multiple times to include compounds from outside the plant kingdom, as well as those that share a biosynthetic origin with alkaloids but do not have a basic nitrogen. 16,17In the broadest sense, alkaloids are nitrogencontaining compounds derived from secondary, or specialised, metabolism.However, it is useful to categorise alkaloids further, not with the intention of excluding compounds, but to help comprehend similarities and differences in the compounds' origins (Fig. 2).The true alkaloids, referred to as complex alkaloids in this review, are compounds in which the nitrogen atom is derived from an amino acid and is part of a heterocycle.Proto-alkaloids, or simple alkaloids, are amines derived from amino acids but do not have a nitrogen heterocycle.Pseudo-alkaloids are nitrogen-containing metabolites in which the nitrogen is introduced at a late stage through an enzymatic process such as transamination.

Major alkaloid classes
This review focusses on complex 'true' alkaloids from plants: compounds derived from an amino acid containing a nitrogen heterocycle.Despite their wide structural diversity  Benjamin is currently a UKRI Future Leaders Fellow and manages a research group investigating the origins and applications of plant natural product biosynthesis.and taxonomic distribution, the biosynthetic origins of these compounds follows a unifying chemical logic centred around the formation and reactivity of the iminium cation. 18I will examine the rst steps of alkaloid biosynthetic pathways: the transition from primary to specialised metabolism and formation of the dening scaffold.The metabolic, enzymatic and evolutionary origins of these steps will be reviewed, including the latest developments in the eld.
This review will cover all major complex alkaloid families in plants.Compounds derived from tyrosine include benzylisoquinolines, tetrahydroisoquinolines and the Amaryllidaceae alkaloids.Although not classically described as alkaloids, the tyrosine-derived betalains share many biosynthetic features with alkaloids and will be discussed.The tryptophan-derived monoterpene indole alkaloids are included.The anthranilate derived acridone and quinoline alkaloids are only briey mentioned, as they do not subscribe to the biosynthetic patterns described in this review.Polyamine derived alkaloids including piperideine, tropane, quinolizidine, pyrrolizidine and Nicotiana alkaloids are all included.It is hoped that the principles of biosynthesis discussed in this review can be applied to many alkaloid families not directly mentioned.

Patterns in biosynthesis
The rst steps in an alkaloid pathway are the most crucial, acting as the gateway to a new chemical space.They are important metabolically, as they direct ux away from primary and into specialised metabolism.They are signicant chemically and enzymatically as they involve the formation of a new molecular structure.They also play a foundational role in the evolution of the pathways.
There are four steps that are typically present in the rst steps of complex alkaloid biosynthesis: (i) accumulation of an amine precursor, (ii) accumulation of an aldehyde precursor, (iii) formation of an iminium cation and (iv) a Mannich-like reaction (Scheme 1).This nal step is oen considered the "scaffoldforming", signature, or rst-committed step into a pathway.
A major origin of variation in these generalised steps is whether they are intermolecular or intramolecular (Table 1).For example, polyamine-derived alkaloids generally form a cyclic iminium through intramolecular condensation, followed by an intermolecular Mannich-like reaction.In contrast, the Pictet-Spengler step that contributes to major alkaloid families, such as the benzylisoquinolines, is an intermolecular condensation, involving formation of an iminium intermediate, followed immediately by an intramolecular Mannich-like reaction.
1][22][23][24][25][26][27][28][29][30][31][32][33][34] In order to differentiate this review, and to highlight its focus on the patterns in the early steps of biosynthesis, it will not be structured by alkaloid class but instead by the particular step in the aforementioned biosynthetic model (Scheme 1).By viewing alkaloid biosynthesis in this holistic manner, it may be possible to gain insights from one pathway that can be applied to aid elucidation of another.

Amine accumulation
The nitrogen in complex alkaloids originates from amino acid metabolism.The majority of alkaloids do not directly incorporate an amino acid, but instead use a primary amine derivative.6][37][38] In the general pattern of alkaloid biosynthesis proposed in this review, amines accumulate beyond typical concentrations to enable ux to be directed into alkaloid metabolism without disruption to existing pathways.This usually requires changes to metabolism bought about by gene duplication.The contribution of primary metabolism to plant chemical diversity has been recently reviewed. 39or essential or abundant amines, such as putrescine or tyramine, accumulation may result from higher expression or duplication of a pre-existing gene without modication to the enzyme's substrate scope.Accumulation of amines that are typically at low concentrations or absent, such as cadaverine or homospermidine, may require the evolution of new enzyme activity through modication of substrate scope. 40Other possible routes to the accumulation of amines include boosting the concentration of amino acid precursors, or reducing degradative catabolic pathways.The amines contributing to alkaloid biosynthesis can be split into two categories: polyamines, derived from lysine, arginine and ornithine; and aromatic amines, derived from tryptophan and tyrosine.

Polyamines
The polyamines putrescine, spermine and spermidine play essential roles in plants, for example in seed development 41 and protein translation 35,42,43 (Fig. 3).Cadaverine is found only in selected plant taxa (e.g.legumes) and contributes to stress responses. 44The simple polyamines putrescine and cadaverine are precursors to major alkaloid classes containing pyrrolinium or piperideine moieties respectively.The triamine dimer of putrescine, homospermidine, is a precursor to the pyrrolizidine alkaloids.2.1.1.Putrescine.Putrescine is incorporated into alkaloids via the intermediate N-methylpyrrolinium (Scheme 2A).In plants, putrescine can be derived from arginine or ornithine (Scheme 2B).Ornithine decarboxylation is the typical route in eukaryotes. 43Ornithine decarboxylases (OrnDCs) have been identied and characterised from nicotine 45 and tropane alkaloid 46,47 producing plants.An alternative route to putrescine proceeds via arginine.In some plants, such as Arabidopsis, OrnDC is absent and the arginine pathway is the sole route to putrescine. 48The arginine pathway starts with decarboxylation of arginine catalysed by arginine decarboxylase, 47,48 and appears to have been derived from the endosymbiont precursor to the chloroplast. 49,50he nightshade family (Solanaceae) includes species rich in putrescine-derived alkaloids, such as Nicotiana tobaccum (nicotine) and tropane alkaloid producers Atropa belladonna and Solanum sp.In this family, the ornithine pathway appears to be the major source of putrescine for alkaloid biosynthesis. 51,52A number of Solanaceae species contain two copies of OrnDC, originating from a putative duplication event prior to the emergence of the family. 53In Nicotiana, one OrnDC coexpresses with alkaloid biosynthesis genes in the roots. 53The duplication of OrnDC may have led to tissue specic accumulation of putrescine, ultimately enabling the formation of diverse putrescine-derived alkaloids in Solanaceous plants.
2.1.2.Cadaverine.Cadaverine is a precursor to the piperideine moiety that is incorporated into alkaloids including the granatane, lycopodium and quinolizidine families (Scheme 3A).Cadaverine is derived from lysine by decarboxylation (Scheme 3B).Bifunctional lysine/ornithine-decarboxylases (Lys/OrnDCs) have been characterised from quinolizidine and lycopodium alkaloid producers. 54,55Lys/OrnDCs are homologous to OrnDCs, but demonstrate enhanced lysine decarboxylase activity.The switch from an OrnDC to a bifunctional Lys/OrnDC appears to have occurred independently at least twice: within the legumes (Fabaceae), preceding the origin of quinolizidine alkaloid biosynthesis, and in the lycophyte lineage, preceding the origin of the lycopodium alkaloid pathway. 55,56Both shis in activity occurred with a substitution from histidine-344 to tyrosine or phenylalanine, enhancing activity with lysine. 54,55Plants with Lys/OrnDC do not appear to retain a separate OrnDC paralog, indicating gene duplication of OrnDC is not necessary for the emergence of Lys/OrnDCs.This may be because Lys/OrnDCs maintain sufficient OrnDC activity, or because the alternative arginine pathway fulls the plants' requirement for putrescine.
2.1.3.Homospermidine.The pyrrolizidine alkaloids are toxic alkaloids present in multiple families including Apocynaceae, Asteraceae and Boraginaceae. 28,58The core pyrrolizidine moiety (also known as the necine base) is derived from homospermidine (Scheme 4A), which is formed from putrescine and spermidine in a reaction catalysed by homospermidine synthase (Scheme 4B).Spermidine is essential for normal plant growth, 35,59 and is formed by spermidine synthase from the substrates putrescine and S-adenosylmethioninamine, a decarboxylated form of the co-factor Sadenosyl methionine (SAM).
Homospermidine synthase (HSS) has evolved from deoxyhypusine synthase (DHS) on at least six independent occasions. 42,58,60,61Deoxyhypusine synthase has a key role in eukaryotic translation, modifying the initiation factor eIF5A with an aminobutyl group derived from spermidine (Scheme 4C).It is also able to accept putrescine as a substrate in place of eIF5A.Duplications and neofunctionalisations have led to enhancement of putrescine activity and reduction of eIF5A activity to yield HSS. 61 Convergence is also evident on the residue level, with identical active site substitutions occurring in each independent HSS origins. 62

Aromatic amines
The aromatic amino acids tyrosine and tryptophan are precursors to major alkaloid families, typically via their primary amine derivatives, tyramine and tryptamine.Simple tyramine and tryptamine-derived products are ubiquitous in green plants (Fig. 4).For example, tyramine contributes to the structure of suberin, a cell wall biopolymer, 37 and hordenine, found in barley, is a derivative of tyramine with anti-fungal properties. 38ryptophan contributes to a number of simple proto-alkaloids in plants via indole including indole-3-acetic acid and the indigo precursor indican.Tryptamine is a precursor to serotonin and psychoactive proto-alkaloids such as dimethyltryptamine. 36yramine and tryptamine are derived from tyrosine and tryptophan by decarboxylation, catalysed respectively by tyrosine decarboxylase (TyrDC) 63 and tryptophan decarboxylase (TrpDC). 64,65These decarboxylases are ubiquitous in green Scheme 5 Tyrosine derived alkaloids.Portion from tyrosine highlighted in blue.plants: TyrDCs and TrpDCs form distinct clades in the plant aromatic amino acid decarboxylase (AAAD) protein family; both appear to have originated in the angiosperm ancestor. 66Despite their evolutionary distance, they share active site and substrate recognition sites: substitution of a single residue in Papaver somniferum TyrDC with the equivalent amino acid from Catharanthus roseus TrpDC (S372G) enables TyrDC to accept indolic substrates, and the complementary mutation in CrTrpDC has the equivalent effect, allowing it to accept phenolic substrates. 672.1.Tyrosine.Tyrosine is the precursor to multiple alkaloid families including the benzylisoquinolines (BIA), the Amaryllidaceae alkaloids and the betalains.A TyrDC from Amaryllidaceae alkaloids biosynthesis has recently been discovered and enables incorporation of tyramine into the structures.68 Many alkaloids are derived from dopamine, for example the BIAs and alkaloids from Colchicaceae, Erythrina and ipecac (Carapichea ipecacuanha) (Scheme 5).Dopamine can be formed from tyrosine through two steps, hydroxylation and decarboxylation (Scheme 6).24 Two TyrDCs were identied in the BIA producing Papaver somniferum, both able catalyse decarboxylation of L-DOPA and L-tyrosine, with a slight preference in vitro for L-DOPA.[69][70][71] However, the BIA associated enzyme catalysing the hydroxylation step remains unknown.The 3-hydroxy group introduced by this missing hydroxylase is essential for downstream Pictet-Spengler reactions (see Section 5.2).72,73 In the BIA producing Papaver species, dopamine accumulates to a high concentration ($16% cellular dry weight of P. bracteatum) and is stored in the vacuole.74,75 Tyramine accumulates up to 5 mM in the latex of a low-alkaloid variety of P. somniferum.76 These observations indicate that tyrosine derived amines accumulate in BIA producing plants; however, the metabolic and evolutionary origins of this accumulation remain unclear.
By contrast, the metabolic and evolutionary origins of tyrosine accumulation in betalain producing plants is well characterised.Betalain pigments are derived from tyrosine, but unlike typical alkaline alkaloids, they incorporate amino acids directly without decarboxylation.The non-proteinogenic amino acids L- DOPA and cyclo-DOPA are precursors to betalains, and are formed through hydroxylation and oxidative cyclisation of tyrosine catalysed by cytochrome P450s in the CYP76AD1-subfamily. 77The hydroxylation is equivalent to the missing step in BIA dopamine biosynthesis.The CYP76AD1-subfamily in the betalain producing Caryophyllales form two clades, 78 with enzymes in the b-clade (CYP76AD5/6/15) catalysing the hydroxylation of tyrosine, 79,80 and enzymes in the a-clade (CYP76AD1-4) capable of catalysing both tyrosine hydroxylation and L-DOPA oxidative cyclisation to form cyclo-DOPA. [79][80][81][82] As betacyanins are derived from cyclo-DOPA whereas betaxanthins are not, the ratio of aand b-clade enzymes can determine the ratio of betaxanthins and betacyanins (see Section 4.2.2). 83 The metabolic origins of the betalain pathway lie in primary metabolism and tyrosine biosynthesis. 84Arogenate dehydrogenases (AroDH) catalyse the formation of tyrosine and typically have strong product feedback inhibition. 85In the Caryophyllales, there are two clades of AroDH paralogs: AroDHbs, which have the typical tyrosine sensitivity, and AroDHas, which show relaxed sensitivity to tyrosine.These two clades emerged in the ancestor of the core Caryophyllales, prior to the emergence of betalain biosynthesis: AroDHas may cause accumulation of tyrosine required for betalain biosynthesis.This shows how changes in primary metabolism resulting in the Scheme 9 Metabolite fate of putrescine.Typically, putrescine is converted to GABA and shuttled into the citric acid cycle.In plants producing putrescine-derived alkaloids, a portion of putrescine is shuttled into alkaloid biosynthesis through methylation and oxidation.
Scheme 8 Biosynthetic origins of acridone and quinoline alkaloids.Anthrinilate synthase is typically inhibited by tryptophan, but in acridone/ quinoline alkaloid producing plants a second anthrinilate synthase is present lacking sensitivity to tryptophan.
accumulation of metabolites may have led to the evolution of new pathways.
2.2.2.Tryptophan.Tryptamine, the amine derivative of tryptophan, is a precursor to b-carboline alkaloids including the harmala alkaloids, nigakinone, and, most notably, the monoterpene indole alkaloids (MIA) (Scheme 7). 86MIA producing species in the Gentianales order, including Catharanthus roseus, appear to have a single copy of TrpDC. 64,87,88However, the camptothecin producing Camptotheca acuminata (Cornales) has two copies of TrpDC, one of which is regulated developmentally and the other which is elicited by stress responses; 89 this duplication may have contributed to the accumulation of tryptamine in this species.
The acridone and quinoline alkaloids, derived from the tryptophan precursor anthranilate, do not follow the typical alkaloid biosynthetic pattern described in this review. 90owever, their origins are informative regarding how precursors can accumulate (Scheme 8).Anthranilate is formed from chorismate through a reaction catalysed by anthranilate synthase (AS), which is typically feedback inhibited by tryptophan, enabling regulation of the tryptophan accumulation. 91The acridone alkaloid producing species Ruta graveolens has two copies of the ASa subunit: ASa2 is constituently expressed and inhibited by tryptophan, whereas ASa1 is upregulated upon elicitation and has reduced sensitivity to tryptophan. 92,93ASa1 enables the accumulation of anthranilate that ultimately leads to increased alkaloid biosynthesis.

Summary
The nitrogen atom denes alkaloids.Therefore understanding how nitrogen is channelled into alkaloid biosynthesis is vital for our comprehension of their metabolic and evolutionary origins.Many of the amine precursors of complex alkaloids are present in the majority of plant taxa.Modications to the metabolism of these precursors can result in their accumulation and channelling into alkaloid biosynthesis.In both betalain and acridone biosynthesis core metabolic enzymes have been duplicated, and their feedback inhibition reduced, enabling accumulation of precursors. 84,93In the Solanaceae, duplication of OrnDC may have led to the accumulation of putrescine. 53n pathways where the amine precursor is not part of primary metabolism, enzyme evolution has occurred.This includes the repeated evolution of HSS from DHS in pyrrolizidine alkaloid biosynthesis, 58 and of Lys/OrnDC from OrnDC for piperideine alkaloid formation. 55These events have happened multiple times independently due to the inherent promiscuity of the ancestral enzymes.
Does accumulation of amine precursors presage alkaloid evolution?Or does modication to primary metabolism serve to push ux through a pre-existing pathway?Phylogenetic analyses of genes involved in the formation of amine precursors in betalain (AroDH), piperideine (Lys/OrnDC) and pyrrolinium biosynthesis (OrnDC) place enzyme duplication/evolution events prior to the emergence of alkaloid producing taxa. 53,55,84hilst further verication is needed of this chronology, it suggests that amine accumulation pregures the emergence of alkaloid biosynthesis.

Aldehyde accumulation
Complex alkaloid biosynthesis typically requires an aldehyde precursor.The aldehyde must accumulate to a high concentration and in close proximity to the amine precursor to enable the next stage of general alkaloid biosynthesis, formation of a reactive iminium intermediate.For polyamine-derived alkaloids, this aldehyde is formed through oxidative deamination of a terminal amine on the polyamine precursor, forming an amino aldehyde.In aromatic amine derived alkaloids, the aldehyde moiety is present on a distinct molecule, typically a secondary metabolite.

Amino aldehydes
The oxidative deamination of a primary amine on a polyamine produces an amino aldehyde.As described below (Section 4.1), this leads to the spontaneous intramolecular formation of a cyclic iminium, which is a key electrophilic intermediate in alkaloid biosynthesis.The catabolism of polyamines in primary metabolism involves oxidative deamination catalysed by copper-containing amine oxidases (CuAOs) or FAD-dependent polyamine oxidases. 94The enzymes responsible for aldehyde formation in polyamine derived alkaloid biosynthesis appears to have been derived from these catabolic enzymes.iminium precursor N-methylpyrrolinium.This is derived from putrescine via a methylation to N-methylputrescine, catalysed by putrescine N-methyltransferase (PMT), 96 and oxidative deamination, catalysed by N-methylputrescine oxidase (MPO) (Scheme 9). 97,98The product, N-methylaminobutanal, spontaneously condenses to form N-methylpyrrolinium. The biosynthesis of N-methylpyrrolinium has been investigated primarily in the Solanaceae (e.g.Nicotiana), 22,53 though the taxonomic distribution of N-methylpyrrolinium alkaloids indicates there are multiple independently evolved pathways. 99MTs are SAM-dependent methyltransferases that evolved from spermidine synthases (SPDSs). 100,101Wild-type SPDSs demonstrate PMT activity, indicating that neofunctionalisation of SPDS to PMT can occur with ease. 101The PMT involved in tropane and nicotine biosynthesis emerged from a SPDS duplication in the Solanales lineage, prior to the origin of the Solanaceae. 53,101PO is a copper-containing amine oxidase localised to the peroxisome. 95The Solanaceae MPO originated from a CuAO homolog generated by a whole genome triplication event. 53,95As part of polyamine catabolism, peroxisomal CuAOs oxidise putrescine to 4-aminobutanal, which is then directed into the TCA cycle via 4-aminobutyrate and succinate (Scheme 9). 102,103It is possible that the methyl group introduced by PMT prevents recognition by catabolic enzymes, allowing N-methylaminobutanal to accumulate.

Amino acid origins
Aldehydes that are incorporated into amino-acid derived alkaloids accumulate independently of the amine precursor.Oen, the aldehyde is a specialised metabolite, already present at high Scheme 12 Biosynthesis of betalamic acid.concentration.Alternatively, aldehyde accumulation could be triggered by gene duplications or other changes to metabolism.
3.2.1.Phenylacetaldehydes. BIAs are formed from dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA), both derivatives of tyrosine. 23Recently, a deviation to this route has been found: Macleaya cordata appears to form BIAs via 3,4-dihydroxyphenylacetaldehyde and not 4-HPAA. 1074-HPAA biosynthesis requires two steps from tyrosine, transamination and decarboxylation (Scheme 11).A tyrosine aminotransferase from Papaver somniferum was found to generate 4-hydroxyphenylpyruvate, 108 but the enzyme catalysing the following decarboxylation is unknown.Multiple aromatic acetaldehyde synthases (AASs) catalysing the formation of aldehydes from aromatic amino acids have been identied in plants, 66,109 including an 4-HPAA synthase from salidroside biosynthesis in Rhodiola rosea. 63AASs have evolved convergently from decarboxylases in the PLP-dependent L-amino acid decarboxylase (AAAD) family and represent an alternative candidate for this elusive step in BIA producing species. 66It is noteworthy that in BIA biosynthesis, both amine and aldehyde are derived from tyrosine-this points to the pathway emerging in a tyrosine-rich context.
Caryophyllales DODA a-clade, which split from the nonactive b-clade prior to the formation of betalain pigmentation. 78Various lineages of the DODA a-clade then duplicated and acquired high DODA activity through convergent evolution. 31Seven amino acid residues have been identied that are sufficient to switch a marginally active DODAa into an active DODAa, 113 and there is evidence that the polyphyletic highly active DODAs converged on similar residues at these key positions. 114Analysis of DODA indicates multiple origins of betalain pigmentation in the Caryophyllales, within a context where L-DOPA was accumulating and a DODAa enzyme with low but notable activity was present.As described above (Section 2.2.1), duplication of AroDH led to high tyrosine levels in Caryophyllales.This, in turn, may have enabled generation and accumulation of betalamic acid.
3.3.2.Dihydrocinnamaldehyes.The phenylpropanoid pathway is a likely origin of the aldehydes required to form phenyl-ethylisoquinolines such as those in Cephalotaxus, Schelhammera and Colchicum.The aldehydes contributing to these pathways are likely to be substituted dihydrocinnamaldehydes, [117][118][119] which can be derived from substituted cinnamic acid, by reduction of both the a,b doublebond and carboxylic acid (Scheme 14).The double bond reduction may occur on the CoA-ester bound form of the acid 120 or the free aldehyde. 121

Secologanin
Iridoids are a widespread class of secondary metabolites derived from the monoterpene pathway. 122Within the plant kingdom, iridoids are largely restricted to the Asterids, 123 where they typically occur as glycosides providing chemical defence against biting herbivores. 124Seco-iridoids, such as secologanin, are a subset of iridoids where the 5-membered carbon ring has been oxidatively cleaved.These seco-iridoids are found in multiple plants where alkaloids are absent. 125,126In C. roseus (Gentianales), secologanin is the precursor to all monoterpene indole alkaloids (MIAs).][129] The iridoid biosynthetic pathway has been elucidated in C. roseus, and derives from geraniol (Scheme 15B). 122Secologanin is formed from loganin through the action of secologanin synthase (SLS, CYP72A1). 130SLS is closely related to another enzyme involved in iridoid biosynthesis, 7-deoxyloganic acid hydroxylase (CYP72A224), indicating that substrate recognition is key to CYP evolution. 131C. roseus contains at least two copies of SLS. 132he MIA pathway in Camptotheca acuminata (Cornales) proceeds via secologanic acid and not secologanin. 133Consequently, strictosidinic acid and not strictosidine is the rst MIA.Cytochrome P450s (CYP72A565 and CYP72A610) from C. acuminata are able to catalyse both the hydroxylation 7-deoxyloganic acid to form loganic acid and the oxidative cleavage of loganic acid to form secologanic acid. 134These are closely related to secologanin synthase (CYP72A1) and 7-deoxyloganic acid hydroxylase (CYP72A224).
Nothapodytes nimmoniana (Icacinales) also produces camptothecin, though is more closely related to Gentianales than Cornales, and the pathway appears to proceed using the typical Gentianales route via secologanin. 135Icaninales and Gentianales may share an MIA origin, and this is supported by characterisation of an active secologanin synthase from N. nimmoniana (NnCYP72A1). 136

Iminium formation
A primary amine can condense reversibly with an aldehyde to form an electrophilic iminium cation, also known as a Schiff base (Scheme 16).8][139] The position of the equilibrium can shi towards the products if the iminium is cyclic or conjugated. 140,141urprisingly, the ratio of neutral imine to charged iminium in solution is largely dependent on the pK a H of the amine precursor ($9-10), rather than the pK a H of the iminium itself ($7), meaning that at physiologically relevant pHs the reactive iminium will be more abundant than the imine. 137Iminiums are highly electrophilic and form rapidly, so even when they not at high concentrations, the product of their reaction with a nucleophile can accumulate, provided the reaction is irreversible.This reaction of an iminium with a nucleophile is described here as a Mannich-like reaction, which quenches the reactivity of both the iminium and nucleophile.
Enzyme catalysed formation of iminiums is oen futile due to their high reactivity in water: no enzymes catalysing only iminium formation have been described.However, iminiums can be enzyme substrates, such as in imine reductases, which typically take preformed iminiums (typically conjugated or cyclic) from solution as substrates. 140Yet, iminium formation can be enzyme catalysed when coupled directly to a quenching reaction, such as reduction. 142

Intramolecular
The electrophilic intermediates in polyamine derived alkaloid biosynthesis are cyclic iminiums such as N-methylpyrollinium and D1-piperideine, formed from linear amino aldehydes through intramolecular condensation (Scheme 17).These rings form spontaneously, without the requirement for catalysis, as demonstrated by chemical syntheses. 143,144Even in water, the cyclic iminium will be abundant.The CuAO enzymes forming the amino aldehyde are localised to the peroxisome, and the cyclic iminiums will form upon ejection into the solvent or possibly in the enzyme active site. 95,105

Intermolecular
In the biosynthesis of alkaloids derived from aromatic amines, the iminium intermediate is formed through an intermolecular condensation and is non-cyclic.Typically, these iminiums are at very low concentration in water.This potentially unfavourable equilibrium can be overcome through rapid quenching of the transient iminium through an intramolecular nucleophile, as seen in Pictet-Spengler reactions (see Section 5.2); or through formation of a highly stable conjugated iminium as demonstrated in betalain biosynthesis (Section 4.2.2).
4.2.1.Coupled reactions.In Pictet-Spengler reactions, the scaffold-forming step in the BIA and MIA biosynthetic pathways, the reversible formation of the electrophilic iminium is followed by an irreversible rate-determining intramolecular electrophilic aromatic substitution (Section 5.2). 145,146This essentially couples iminium formation and a Mannich-like reaction; the iminium exists as a transient intermediate.Enzymes catalysing the Pictet-Spengler reactions, norcoclaurine synthase (NCS) and strictosidine synthase (STS), appear to catalyse iminium formation as part of their mechanism. 146,147However, they may also be able to accept pre-formed iminiums, as demonstrated by their ability to bind secondary amine mimics of reaction intermediates. 148,149In Amaryllidaceae alkaloid biosynthesis, the iminium intermediate, norcraugosodine, is conjugated, giving it greater stability in aqueous conditions (Fig. 5). 27,141The enzyme catalysing the subsequent reduction step has not yet been characterised so the nature of enzyme involvement in iminium formation remains unknown.In any reaction requiring intermolecular iminium formation, co-localisation of amine and aldehyde is required.Further details about Pictet-Spenglerases, norbelladine synthase and co-localisation of substrates is discussed in Section 5.2.

Stable iminium.
Betalain biosynthesis is unique amongst all the alkaloids described here as it lacks a Mannichlike step.Instead, the iminium formed from condensation of betalamic acid and an amine/amino acid is stable in water and can accumulate.Iminium stability is conferred by the highly conjugated structure, which also endows the molecules with their pigmentation properties (Scheme 18).
Betalains are all derived from the aldehyde containing betalamic acid, but can be divided into two categories depending on the amine donor: the red betacyanins are derived from cyclo-DOPA, and the yellow betaxanthins are derived from amino acids or other amines (e.g.dopamine, tyramine) (Scheme 18).The iminium formation step appears to occur spontaneously and does not require enzyme catalysis. 150The specic betalain formed therefore appears to depend on the concentrations of amines present.For example, in plants expressing betalain biosynthesis genes heterologously, the ratio of betacyanins and betaxanthins can be controlled solely by varying the amount of the L-DOPA oxidase activity present (see Section 2.2.1).This enzyme forms cyclo-DOPA, and its presence enhances betacyanins at the expense of betaxanthins. 83ike many other pigments, betalains are stored in the vacuole.However, the key enzymes required for cyclo-DOPA and betalamic acid biosynthesis are cytoplasmic. 151  one possible location for iminium formation: the reaction may be promoted in acidic conditions, and betalain selectivity could be controlled by the accumulation of specic amines by tonoplast (vacuolar) transporters.Identication and characterisation of tonoplast transporters in betalain producers would help resolve this question.

Mannich-like reaction
The key step in many alkaloid biosynthetic pathways is a Mannich-like reaction, in which the iminium reacts irreversibly with nucleophile.This reaction quenches reactive species and causes the formation of a new carbon-carbon bond; it is therefore very energetically favourable, to the extent that in many cases the reaction can proceed without enzyme catalysis.
This reaction can be considered the rst committed step into an alkaloid pathway, as the specic combination of electrophile and nucleophile denes alkaloid subtype and is irreversible.Furthermore, it is typically the scaffold-forming step and establishes a heterocyclic structure that is central to alkaloid identity, with impacts on downstream reactions and compound bioactivities.

Polyamine-derived
In polyamine-derived alkaloids, the Mannich-like reaction is typically intermolecular.The nucleophiles are of diverse origin and give rise to specic alkaloid sub-types.These nucleophiles must accumulate in the same subcellular location as the iminium.As the accumulating non-amine component of the alkaloid, they are equivalent to aldehydes required in aromatic amino acid derived alkaloids.
To date, no enzymes have been described that catalyse the intermolecular Mannich-like reactions of alkaloid biosynthesis, and it is possible that the in planta reactions occur spontaneously with no enzyme catalysis.The intermolecular nature of the reaction has led to a modular organisation of in polyamine alkaloid biosynthesis, where a range of different nucleophiles may combine with either N-methylpyrollinium or D1-piperideine to generate chemical diversity.
5.1.1.Polyketide.The best understood Mannich-like reaction in polyamine-derived alkaloid biosynthesis is from tropane alkaloid biosynthesis in the Solanaceae (Scheme 19A).N-Methylpyrollinium is attacked by an enol form of 3-oxoglutaric acid, a nucleophile derived from polyketide biosynthesis.][154] Pyrrolidine ketide synthase (PYKS), a type III polyketide synthase, catalyses the formation of 3-oxoglutaric acid from two malonyl-CoA units.PYKS has an active site with the typical type-III PKS catalytic triad for substrate loading and chain extension (Cys166, His305 and Asn338), but has additional residues (Arg-134 and Ser-340) which limit the enzyme to a single extension step by interacting with the substrate carboxylate moiety. 153hen incubated with PYKS, N-methylpyrollinium and malonyl-CoA form 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid, a key intermediate.spontaneously reacts with N-methylpyrollinium through a decarboxylative Mannich condensation. 153PYKS does not utilise N-methylpyrollinium as a starter unit, nor does the enzyme catalyse the condensation reaction.This observation is supported by the racemic nature of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid, and by the presence of the side products hygrine and cuscohygrine, which may be formed through decarboxylation or a second decarboxylative Mannich condensation respectively. 153he 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid intermediate can be converted to tropinone through the action of tropinone synthase (CYP82M3). 152The mechanism of this is unknown, but a possible route is formation of a pyrollinium cation through hydroxylation and dehydration (Scheme 19B).This intermediate then undergoes a further, possibly spontaneous, intramolecular decarboxylative Mannich condensation to yield tropinone.
Tropane alkaloids emerged independently Erythroxylaceae and Solanaceae. 99However, in Erythroxylum coca (Erythroxylaceae) alkaloids, the tropane ring has an additional carboxymethyl group.This could arise if the carboxylic acid moiety of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid is methylated prior to cyclisation (Scheme 19). 152The enzymes catalysing the formation of tropane alkaloids in the Erythroxylaceae are yet to be fully described. 155e solution of the key step in tropane biosynthesis has implications for other pathways.The 3-oxoglutaric acid nucleophile can also react with D1-piperideine, leading to a set of homologous compounds and pathways to those derived from Nmethylpyrollinium.For example, quenching of D1-piperideine by 3-oxoglutaric acid through a decarboxylative Mannich reaction yields oxo-2-piperidine-butanoic acid.This intermediate could decarboxylate to yield pelletierine, a homolog of hygrine, or undergo oxidative cyclisation to yield norpseudopelletierine, a homolog of tropinone (Scheme 20A). 22,153Pseudopelletierine is a precursor to the granatane alkaloids.The biosynthesis of lycopodium alkaloids such as huperzine is likely to proceed via aldol addition of oxo-2-piperidine-butanoic acid to pelletierine (Scheme 20B). 57,156.1.2.Nicotiana.Alkaloid biosynthesis in Nicotiana has a modular arrangement: a single nicotinic acid-like nucleophile can quench N-methylpyrollinium to form nicotine, or D1piperideine to form anabasine (Scheme 21A).The nicotinic acid-like nucleophile is derived from a nicotinamide co-factorlike pathway that emerged from gene duplications in the Nicotiana lineage (aer the split of Solanum).Interestingly, these gene duplications were coupled with duplication of transcription factors and changes to transcription-factor binding sites in genes, ultimately leading to root-specic expression of the nicotine biosynthetic pathway. 53heme 20 Hypothesised formation of piperideine alkaloids.(A) Proposed formation of piperideine derived alkaloids based on known formation of tropane alkaloids.(B) Possible route to lycopodium alkaloids such as huperzine. 57 gene encoding an NADPH-dependent reductase, A622, has a major role in activating nicotinic acid, however neither the substrate nor product structure is known.157,158 It is hypothesised that the nucleophilic compound attacking N-methylpyrollinium or D1-piperideine may be 3,6-dihydronicotinic acid or 3,6-dihydropyridine (Scheme 21B).159 The nal step in nicotine biosynthesis is catalysed by vacuolar located avin-containing oxidases with similarity to berberine-bridge enzymes (BBLs).160 Knocking out all six BBL paralogs in tobacco results in a nicotine-free plant.161 Knockingdown BBL expression results in the accumulation of a reduced nicotine metabolite (dihydrometanicotine), indicating that the enzyme acts aer the condensation step, oxidising the pyridine ring (Scheme 21B).19,160 A similar oxidation is likely to be involved in anabasine production.
It is possible that, like the quenching step in tropane alkaloid biosynthesis, the reaction between N-methylpyrollinium or D1-piperideine and the nicotinic acid derivative is spontaneous.This is supported by the observation that the enantiomeric excess of the (S)-enantiomer in natural nicotine is caused by enantioselective enzymatic demethylation of (R)-nicotine to (R)-nornicotine and not selective cyclisation. 162The increase in anabasine to nicotine ratio through increased concentration of precursors could also be indication of this. 163Based on the location of BBL it is possible the Mannich-like reaction occurs in the vacuole.
5.1.3.Dimerisation.In the scaffold-forming steps of quinolizidine alkaloid biosynthesis, both the nucleophile and electrophile are derived from D1-piperideine.The dimerization between the two monomers requires an imine-enamine tautomerisation followed by Mannich condensation (Scheme 22A).No enzymes catalysing these reactions have been described.In solution, D1-piperideine interconverts between multiple species at different pHs, including a-tripiperideine and the hydroanabasine dimer (Scheme 22B). 143,164In plants, quinolizidines do not always co-occur with D1-piperideine derived alkaloids, indicating that dimerization of D1-piperideine must be sufficiently slow to allow quenching by other nucleophiles.
Density functional theory calculations indicate that the dimerization reaction between D1-piperideine and 2-piperideine occurs spontaneously to form the (R,R) or (S,S)-tetrahydroanabasine, but formation of the (R,S)-or (S,R)- tetrahydroanabasine dimers proceeds more slowly. 165The diastereoselectivity of the spontaneous reaction matches the biosynthetic origins of compounds such as (À)-lupinine, a major alkaloid found in Lupinus (Scheme 22C). 32However, quinolizidines are oen found in high enantiomeric excess, suggesting that a stereoselective enzyme catalyses the dimerization step, or downstream enzymes exert enantioselectivity.
5.1.4.Pyrrolizidine formation.The scaffold-forming step of pyrrolizidine alkaloid biosynthesis is unusual in the context of polyamine derived alkaloid biosynthesis in that the Mannichlike reaction is intramolecular (Scheme 23A).This reaction can occur spontaneously and stereoselectively in acidic conditions to yield the trans-product (Scheme 23B). 144In plants, pyrrolizidines typically accumulate as single enantiomers.Furthermore, pyrrolizidines with cis-stereochemistry are common (e.g.nervosines, Scheme 23C). 28These factors point to an enzyme controlled intramolecular Mannich condensation.Alternatively, there may be selective degradation of isomers.Due to the inherent reactivity of the dialdehyde, the enzyme that catalyses oxidative deamination may also control the subsequent stereoselective cyclisation.

Aromatic amino-acid derived
Major classes of alkaloids generated from aromatic amines proceed via a Pictet-Spengler reaction.In this reaction, the intermolecular condensation of an amine and aldehyde forms an iminium, which is then quenched through intramolecular electrophilic aromatic substitution. 166,167The tethered nucleophilic aromatic system bypasses the requirement for accumulation of a distinct nucleophile.Mechanistic and biocatalytic aspects of Pictet-Spenglerases have been reviewed recently. 10he entry to the tyramine derived Amaryllidaceae alkaloids does not involve a Pictet-Spengler reaction; instead, the Mannichlike step is a reduction, in which the hydride can be considered an intermolecular nucleophile.
5.2.1.Tetrahydroisoquinolines.The Pictet-Spengler reaction between dopamine and an aldehyde yields a tetrahydroisoquinoline, and can proceed in aqueous conditions, catalysed by acid 168 or inorganic phosphate. 72Key to the facile nature of this reaction is the dopamine 3-hydroxyl group, which increases the nucleophilicity of the catechol ring and directs the substitution to the 6-position (Scheme 24A).As the reaction has a low energy barrier, the presence of tetrahydroisoquinoline metabolites are no guarantee of a Pictet-Spenglerase catalyst. 169owever, Pictet-Spenglerases may serve to increase the rate of reaction, provide substrate selectivity or determine stereoselectivity.
1][172][173] NCS is a member of the pathogenesisrelated 10/Bet-v1 (PR10) protein family, a diverse set of small (15-25 kDa), soluble proteins notable for their ability to bind diverse ligands. 172,174NCSs from Ranunculaceae (e.g.Thalictrum avum) are single domain proteins, but NCS from the Papaveraceae, which includes opium poppy (P.somniferum), are present as fused repeats, with up to four consecutive domains on the same polypeptide chain. 175Recently, two more PR10s involved in BIA biosynthesis have been identied: thebaine synthase 176 and neopinone isomerase. 177Outside BIA metabolism, PR10s have roles in defence responses 178 and binding intermediates in secondary metabolism pathways. 179,180CS contains an N-terminal signal peptide, which targets the enzyme to the vacuole via the endoplasmic reticulum. 172As previously described, the vacuole may be a site with high dopamine concentrations. 75However, it has been proposed that the primary site of NCS activity is en route to the vacuole, in the lumen of the endoplasmic reticulum, possibly in alkaloid specic vesicles. 23,181The details of subcellular trafficking in BIA biosynthesis including the primary location of NCS activity has not yet been resolved.
The mechanism of NCS (Scheme 24C) has been elucidated through structural 182,183 and computational analyses. 147Dopamine binds in the enzyme active site prior to 4-HPAA, with the catechol 3-OH H-bonding to the Lys-122 residue deep in the active site and the residues Glu-110 and Asp-141 interact with the dopamine amine group. 148Iminium formation probably involves acid/base catalysis from Glu-110 and Asp-141, and may also involve changes to the enzyme structure. 183The key steps of C-C bond formation and subsequent loss of proton are catalysed by Lys-122 and Glu-110 respectively.
To date, all characterised NCS enzymes are from Ranunculales.The control of the Pictet-Spengler reaction in BIA biosynthesis outside this order, such as in sacred lotus (Nelumbo nucifera, Proteales) or the magnoliids, is unknown.NCS activity was found to be widely distributed in plant extracts from across angiosperms, including those not producing BIAs, but the proteins responsible for this were not identied. 171A number of NCS homologs are present in the sacred lotus genome and some feature some key NCS catalytic residues; however, these have not been characterised. 184Phylogenetic analysis of NCSs across published genomes place these lotus sequences away from the NCS clade indicating an independent origin of BIAs in lotus. 185Sacred lotus produces BIAs with both (S)-and (R)-stereoisomers, 186 suggesting that: (i) both (S)and (R)-selective NCSs are present, (ii) there is an epimerisation step in the pathway or (iii) the Pictet-Spengler reaction is not enzyme catalysed.
There are multiple examples of alkaloids derived from dopamine and dihydrocinnamaldehyde derivatives, such as those found in the Colchicaceae (e.g.Colchicum, Schelhammera) and Cephalotaxus (Scheme 25B). 20,119Cryptostylines (Cryptostylis) are products of Pictet-Spengler condensation of dopamine and benzaldehydes (Scheme 25C).To date, no enzymes have been reported catalysing the Pictet-Spengler step in these pathways.
Strictosidine synthases are part of the nucleophilic attack six-bladed b-propeller (N6P) superfamily, which includes enzymes such as paraoxonases and lactonohydrolases, and likely evolved from metal dependent enzymes. 190,191Strictosidine synthase-like (SSL) proteins are ubiquitous in plants, and although their activities are largely unknown, they appear to have diverse functions including in anther development 192 and defence response. 193n plants, SS is localised to the vacuole of laticifer cells. 194,195owever, the enzymes involved in the formation of its substrates, tryptophan decarboxylase and secologanin synthase, are in the cytoplasm and endoplasmic reticulum respectively.Strictosidine glucosidase, the enzyme that converts strictosidine into reactive aldehyde intermediates, is localised to the nucleus. 196Therefore, transportation of substrates and products are necessary.The efflux of strictosidine across the vacuolar membrane (tonoplast) is controlled by CrNPF2.9, a member of the nitrate/peptide transporter family (NPF). 197The transporters involved in the vacuolar import of tryptamine and secologanin are unknown.
Strictosidine synthase (SS) has so far only been characterised from Gentianales.In MIA biosynthesis in Camptotheca acuminata (Cornales), the precursor to MIAs is 3-(S)-strictosidinic acid, 133 but a putative strictosidinic acid synthase has not been identied, and there is no clear ortholog to SS in the transcriptome or genome. 198Based on the substrates employed, it is possible that the MIA pathways in Camptotheca and the Gentianales are convergent, and if so, the Pictet-Spenglerase is unlikely to be orthologous to SS.In contrast, MIA biosynthesis in Nothapodytes nimmoniana (Icaninales) and Gentianales are likely to share a common origin, 136 and accordingly a putative strictosidine synthase homologous to those in Gentianales has been identied in its transcriptome. 199he Harmala alkaloids (Peganum harmala) may be derived from a Pictet-Spengler condensation of a tryptophan-derived amine with pyruvate.However, few details of the biosynthesis have been elucidated. 200.2.3.Reduction.The rst committed step into the Amaryllidaceae alkaloids (AA) is the condensation of trypamine and 3,4-dihydroxbenzaldehyde to form the imine norcraugosodine, followed by reduction to yield norbelladine (Scheme 27A).The reduction step is likely to be catalysed by an NAD(P)H dependent dehydrogenase/reductase. Within the general scheme of alkaloid biosynthesis, hydride can be considered an intermolecular nucleophile quenching the electrophilic iminium.AAs are therefore unique amongst alkaloid pathways in requiring intermolecular reactions for both iminium formation and quenching.It is possible that this enzyme may also catalyse the formation of norcraugosodine, though the conjugated iminium may be sufficiently stable to reach appreciable levels in solution (see Section 4.2.1). 141n NADPH-dependent short chain reductase from N. pseudonarcissus capable of catalysing the formation of norbelladine from trypamine and 3,4-dihydroxbenzaldehyde has been reported. 201However, this enzyme was characterised as noroxomaritidine reductase, as this activity was 400Â faster than the nobelladine synthase activity (Scheme 27B).The low norbelladine synthase activity indicates this is a side reaction and unlikely to be relevant in planta.However, the result shows in principle that norbelladine can be formed by a reductase incubated with tyramine and 3,4-dihydroxybenzaldehyde.
Surprisingly, a homolog of norcoclaurine synthase (NCS), has been proposed to catalyse the formation of norbelladine from tyramine and 3,4-dihydrobenzaldehyde. 202 Whilst it is conceivable that an NCS-type enzyme could aid in iminium formation, it is unlikely to catalyse the subsequent reduction step.Therefore, the key step in AA biosynthesis, the formation of norbelladine, remains unresolved.[205]

Discussion
Numerous alkaloid biosynthetic pathways exist in plants, and many of these follow the pattern described in this review: amine and aldehyde accumulate and condense into an iminium, which is quenched by a nucleophile in a Mannich-like scaffoldforming reaction.This model of alkaloid biosynthesis highlights how plants exploits simple chemical logic to construct complex molecules. 18It also enables us to gain insight into how these pathways may have evolved.

Precursor accumulation
The key requirement for alkaloid biosynthesis is the accumulation, or increased production, of precursors in a location specic manner.Amine accumulation occurs due to changes in amino acid or polyamine metabolism.This can be triggered by gene duplication leading to neofunctionalisation through loss of feedback inhibition, 84 shis in substrate specicity 61 or changes to regulation. 206Complex alkaloids require a second molecule to accumulate.In the case of aromatic amino acid derived alkaloids, this is an aldehyde, whereas for polyaminederived alkaloids, the accumulating compound is a nucleophile.Aldehydes may be generated directly from accumulating amino acid precursors (e.g.betalamic acid, 4-HPAA) or may be co-opted from existing secondary metabolism (e.g.secologanin).Nucleophiles in polyamine-derived alkaloid biosynthesis have a diverse origin, in secondary metabolism (e.g.polyketides) or from duplicated pathways (e.g.nicotinic acid).

Subcellular localisation
To form a specic alkaloid scaffold, the two reacting species must accumulate in the same subcellular compartment.Whist this compartment may differ between alkaloid pathways, possible locations include the peroxisome or vacuole.Accumulating polyamines are oxidised into amino aldehydes by promiscuous peroxisomal CuAOs and form cyclic iminiums.If a nucleophile is transported to or synthesised in the peroxisome they could react with the cyclic iminiums prior to export to the cytoplasm.
The vacuole seems a likely location for alkaloid biosynthesis: accumulating compounds may be sequestered in the vacuole to prevent cellular damage.Aldehydes for example, are cytotoxic due to their ability to cross-link DNA or proteins.Alkaloid precursors and products have been measured at high concentration in the vacuole. 75,207Furthermore, the Pictet-Spengler reaction in MIA biosynthesis occurs in the vacuole 197 and other enzymes involved in key scaffold forming steps in alkaloid biosynthesis have vacuolar targeting sequences (e.g.NCS, BBL). 160,172Reactive specialised metabolites are oen sequestered in the vacuole and its acidic conditions may promote spontaneous reactions. 207,208As the pathway matures and comes under greater regulation, 206 the reaction may migrate to a different subcellular location.

Spontaneous reactions
In a cellular environment, two molecules at high concentration with complementary reactivity may combine spontaneously, without enzyme catalysis.Spontaneous reactions can lead to a leap in chemical complexity, as they are not reliant on existing enzyme mechanisms or substrate constraints.Therefore, new chemical scaffolds can be formed without the evolution of new enzyme activities.
Recently the Mannich-like reaction from tropane alkaloid biosynthesis has been shown to occur without enzyme catalysis. 153In other polyamine-derived alkaloids, evidence for spontaneous quenching can be found in the presence of multiple stereoisomers in planta 162 or in biomimetic reactions. 143,144,164Furthermore, this non-catalysed reaction can account for the modularity of nucleophile and electrophile.An exemplar of the modularity is tropane and nicotine alkaloid biosynthesis in the Solanaceae: both require N-methylpyrollinium but utilise nucleophiles of different origins.Notably, the gene duplications required to cause accumulation of Nmethylpyrollinium appeared prior to the ancestor of the Solanaceae, whilst the genes required for the formation of nicotinic acid-like nucleophile only arose in the Nicotiana genus.Therefore, specic taxa have evolved different 'solutions' to an abundance of N-methylpyrollinium by accumulating different nucleophiles that react with the iminium and form bioactive compounds.Similarly, different electrophiles can also react with the same nucleophile, such as the homologous tropane and granatane alkaloids derived from 3-oxoglutaric acid plus Nmethylpyrollinium or D1-piperideine respectively.
Iminium formation in betalain biosynthesis is spontaneous and does not involve enzyme catalysis. 150The Pictet-Spengler reactions of MIA and BIA are enzyme catalysed, but they have a low activation barrier and can occur in relatively mild aqueous conditions. 72,209With sufficient concentration of precursors, the Pictet-Spengler reaction can occur within a cell without a specic catalyst. 169Therefore, it is conceivable that the Pictet-Spengler step emerged prior to the origins of a corresponding Pictet-Spenglerase.

Enzyme evolution
If the new compound formed from a spontaneous reaction confers an advantage-through the development of new bioactivities or reduction in cellular toxicity-an enzyme may be recruited to enhance its reaction rate or provide stereoselectivity.Therefore, a reaction that occurs spontaneously and rapidly in vitro may still have an associated enzyme in vivo.This has been highlighted recently with the identication of enzymes for steps in BIA biosynthesis previously thought to be spontaneous. 176,177nzymes that have evolved to catalyse a "spontaneous" reaction are somewhat unusual in that they oen evolve from protein families with no obvious connection to the reaction or pathway (e.g.NCS, SS).In structurally related alkaloid pathways with independent origins, enzymes catalysing similar "spontaneous" steps would not be orthologous and could have evolved from a variety of protein starting points: they may not be easy to identify through homology.In this sense these enzymes have similarities to the [4 + 2]-cyclases, which are from diverse protein families. 210t is possible that proteins have evolved to inuence the stereochemical course of the reaction without necessarily catalysing it.Such scaffolding proteins are sometimes referred to as dirigent proteins, and have been identied in lignin 211 and iridoid biosynthesis. 212Although none has been identied in alkaloid biosynthesis to date, they could account for the enantiomeric enrichment observed in polyamine-derived alkaloids.

Pathway evolution
This model of alkaloid biosynthesis suggests that the key factor in the evolution of new alkaloids are modications to existing primary and secondary metabolism, and not the emergence of a new enzyme activity.Scaffold-forming enzymes have been considered crucial for catalysing the rst-committed step into alkaloid pathways.However, at least in the early stages of Scheme 28 Hypothetical biosynthesis of nitrarine from Nitraria.This route highlights how the themes of amine/aldehyde accumulation, iminium formation and Mannich-like reactions are modular and lead to chemical complexity. 164,217lkaloid pathway evolution, such steps may occur without enzyme catalysis.
This ts with a general model of metabolite-enzyme coevolution where enzymes catalysing rate-limiting steps are the rst to be recruited into a new pathway. 213When the scaffoldforming step has a low energy barrier, it will not be ratelimiting.Instead, the upstream steps that determine the concentration of precursors will limit ux, and enzymes boosting these will emerge rst.Even some downstream steps with high energy barriers-such as those catalysed by methyltransferases, cytochrome P450 and dehydrogenases-may be established before an enzyme catalysing the scaffold-forming step emerges.A combination of phylogenetics and biochemistry may be able to interrogate the relative timings of enzyme evolution in a pathway.

Conclusion
Methods for the discovery and characterisation of enzymes from plant specialised metabolism have reached maturity, 4 and we will continue to see rapid progress in the elucidation of alkaloid biosynthesis. 214These discoveries will feed into synthetic biology and metabolic engineering, and consequently, the yields and variety of alkaloids available from heterologous production methods will increase. 12,215Phylogenomic approaches are beginning to reveal the evolutionary origins of specialised metabolism, 216 and with ever increasing sequence data, these can be applied to alkaloid producing plants.Furthermore, investigations into aspects of alkaloid biosynthesis such as how ux is channelled from primary metabolism, 39 how promoters have evolved 206 and how pathways are compartmentalised 197,207 will help reveal how alkaloid biosynthesis is integrated into a complete cellular context.
Alkaloids have fascinated scientists for centuries due to their structural intricacies and profound bioactivities.Despite these complexities, alkaloid formation is largely governed by a simple and modular chemical logic, centred around iminium reactivity (e.g.Scheme 28). 18,164,217The ability of nature to generate functional and diverse molecules from simple physical principles will provide inspiration for chemists and biologists for many decades to come.

Conflicts of interest
There are no conicts to declare.

Fig. 2
Fig. 2 Types of alkaloids.Sparteine and quinine are true alkaloids, derived from lysine and tryptophan respectively.Mescaline is a protoalkaloid.Coniine and aconitine are pseudo-alkaloids, with the nitrogen inserted through transamination.

Fig. 3
Fig. 3 Polyamines in plants.Putrescine, spermidine and spermine are ubiquitous in green plants whereas cadaverine and homospermidine only accumulate in selected taxa.

Fig. 4
Fig. 4 Examples of simple aromatic amines in plants derived from tyrosine and tryptophan.

Scheme 7 Scheme 6
Scheme 7 Tryptophan derived alkaloids.(A) Origin of tryptamine from tryptophan.(B) Alkaloids derived from tryptamine.Portion from tryptophan highlighted in blue.
Scheme 18 Betalain biosynthesis.Iminium formation steps are spontaneous without requiring enzyme catalysis.

Fig. 5
Fig. 5 Stability of iminium intermediates.Predicted trend in iminium intermediates in alkaloid biosynthesis based on conjugation.
Scheme 19 Tropane alkaloid biosynthesis.(A) Outline of tropane alkaloid biosynthesis as elucidated in Solanaceae.Methyltransferase step in Erythroxylum is hypothetical.(B) Hypothetical mechanism of tropinone synthase.Note that a methylester would not decarboxylate, giving rise to methylecgonone.

Scheme 25
Scheme 25 Alkaloids derived from dopamine.(A) Alkaloids derived from dopamine and secologanin.Emetine is found in C. ipecacuanha and is derived from a second Pictet-Spengler reaction.(B) Alkaloids derived from dopamine and dihydrocinnamaldehyde.(C) Alkaloids derived from dopamine and substituted benzaldehyde.

Scheme 27
Scheme 27 Amaryllidaceae alkaloid biosynthesis.(A) Entry point into the Amaryllidaceae alkaloid biosynthesis by condensation and reduction.(B) Reaction catalysed by noroxamartidine reductase.