Something Old, Something New: Conserved Enzymes and the ... · Something Old, Something New: Conserved Enzymes and the Evolution of Novelty in Plant Specialized Metabolism1 Gaurav

Update on Metabolic Evolution

Something Old, Something New: ConservedEnzymes and the Evolution of Novelty in PlantSpecialized Metabolism1

Gaurav D. Moghe and Robert L. Last*

Department of Biochemistry and Molecular Biology (G.D.M., R.L.L.) and Department of Plant Biology (R.L.L.),Michigan State University, East Lansing, Michigan 48824

ORCID IDs: 0000-0002-8761-064X (G.D.M.); 0000-0001-6974-9587 (R.L.L.).

Plants produce hundreds of thousands of small molecules known as specialized metabolites, many of which are of economic andecological importance. This remarkable variety is a consequence of the diversity and rapid evolution of specialized metabolicpathways. These novel biosynthetic pathways originate via gene duplication or by functional divergence of existing genes, andthey subsequently evolve through selection and/or drift. Studies over the past two decades revealed that diverse specializedmetabolic pathways have resulted from the incorporation of primary metabolic enzymes. We discuss examples of enzymerecruitment from primary metabolism and the variety of paths taken by duplicated primary metabolic enzymes towardintegration into specialized metabolism. These examples provide insight into processes by which plant specialized metabolicpathways evolve and suggest approaches to discover enzymes of previously uncharacterized metabolic networks.

The plant kingdom collectively produces hundreds ofthousands of low molecular weight organic moleculestraditionally known as secondary metabolites, some ofwhich have been shown to play roles in abiotic and bioticstress responses (e.g. herbivory defense), beneficial in-sect interactions (e.g. pollinator attraction), and com-munication with other plant and nonplant species (e.g.allelopathy and legume-rhizobia interactions; Saitoand Matsuda, 2010; Pichersky and Lewinsohn, 2011;Wink, 2011). These metabolites have been widely usedthroughout the course of human history as medicines,spices, perfumes, cosmetics, and pest-control agents aswell as in religious and cultural rituals. For the past 150years, there has been a strong focus on documenting thechemical diversity of secondary metabolites in the plantkingdom, leading to the discovery of diverse classes ofcompounds such as terpenes, flavonoids, alkaloids,phenylpropanoids, glucosinolates, and polyketides.These secondary compounds were historically differ-entiated from products of primary metabolism, such assugars, amino acids, nucleic acids, and fatty acids, asbeing nonessential for plant survival (Sachs, 1874;Kossel, 1891; Hartmann, 2008). However, by the 1980s,important functional roles began to be elucidated formetabolites previously classified as secondary, such asthe phenolics (e.g. plant-microbe interactions and UV-Blight protection; Bolton et al., 1986; Peters et al., 1986; Liet al., 1993; Landry et al., 1995), alkaloids (defenseagainst herbivory; Steppuhn et al., 2004), and terpenes

(defense against herbivory, antimicrobial activities, andvolatile pollinator attractants; Papadopoulou et al.,1999; Schiestl and Ayasse, 2001; Erbilgin et al., 2006;Nieuwenhuizen et al., 2009). This change in our un-derstanding of their roles has led to the coining of a newterm, specialized metabolites, for these compounds,both to acknowledge their importance and to reflect thefact that many of them are phylogenetically restricted(Pichersky et al., 2006; Pichersky and Lewinsohn, 2011).

Although the structural diversity of specialized me-tabolites far exceeds that of primary metabolites, all spe-cialized metabolite classes are ultimately derived fromprimary metabolic precursors (Wink, 2011). For example,phenylpropanoids are derived from the amino acid Phe(Vogt, 2010), while the biosynthetic blocks of terpenes,isopentenyl diphosphate and dimethylallyl diphosphate,originate from mevalonate, a sterol precursor, and alter-natively from methylerythritol phosphate, which is de-rived from glycolytic pathway precursors (Kirby andKeasling, 2009). Nitrogen-containing alkaloids are de-rived from a variety of primary metabolites, includingamino acids and purine nucleosides (Facchini, 2001).Over the past 20 years, an increasing number of special-ized metabolic enzymes have also been found to havetheir origins in primary metabolic pathways (Weng,2014). Such shifts in enzyme function are made possibleprimarily by the process of gene duplication, which isvery common in plants.

GENE DUPLICATION: THE DRIVER OFMETABOLIC INNOVATION

The evolution of metabolic pathways has been asubject of study for decades (Jensen, 1976). Pathwaysmay evolve by changes in regulatory and protein-coding

1 This work was supported by the National Science Foundation(grant nos. MCB–1119778 and IOS–1025636).

* Address correspondence to [email protected]/cgi/doi/10.1104/pp.15.00994

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http://orcid.org/0000-0002-8761-064X

http://orcid.org/0000-0001-6974-9587

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.15.00994

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sequences of single-copy genes, gene duplication andsubsequent divergence, and biochemical noise (Wenget al., 2012). These processes may be further influencedby drift and/or selection acting on specific alleles anddriving these alleles to fixation in populations. Geneduplication is a central genetic mechanism for generat-ing novel specialized metabolic enzymes because, aslong as the duplicated gene is not lost, it allows for theconservation of old functions while creating new op-portunities for metabolic diversification.Gene duplication occurs in three ways, tandem,

segmental, and whole-genome duplication, and thegenes originating via these processes can diverge inexpression and amino acid sequence, giving rise to newbiochemical activities. Analysis of genome sequences inmultiple plant species revealed that duplicates of pri-mary metabolic enzyme genes arising out of tandem orwhole-genome duplication have different evolutionaryfates than those encoding duplicates of specializedmetabolic enzymes. For example, genes that participatein primary metabolism, such as those involved in car-bohydrate, lipid, amino acid, and nucleotide metabo-lism, generally tend to revert back to a single-copystatus after tandem duplication, while specializedmetabolic enzyme genes, such as those involved insinapate ester and quercitin production in Arabidopsis(Arabidopsis thaliana) and kaempferol production insoybean (Glycine max), tend to be retained as duplicates(Chae et al., 2014). In contrast, after whole-genomeduplication, both specialized and primary metabolicenzyme genes (e.g. those involved in glucosinolate andlignin biosynthesis and amino acid biosynthesis, re-spectively) tend to return to single-copy status (Chaeet al., 2014). It has been proposed that the retention/lossbiases between primary and specialized metabolismgenes may occur because of a potential need for main-taining gene dosage among primary metabolic genes(Freeling and Thomas, 2006), although other features,such as network connectivity, expression level, breadthof expression, and degree of conservation, may alsoplay an important role (Moghe et al., 2014).The retention of duplicates of genes involved in

specialized metabolism is responsible for several spe-cialized metabolic enzymes, such as terpene synthases(Falara et al., 2011), chalcone synthases/polyketidesynthases (Durbin et al., 1995; Austin and Noel, 2003),and acyltransferases (D’Auria, 2006), being members ofmultigene gene families. An extreme example in thisregard is the plant cytochrome P450 (CYP) gene family,which is estimated to include up to 1% of all genes in avariety of plant species (Mizutani and Ohta, 2010). CYPenzymes are primarily involved in catalyzing oxygen-ation and hydroxylation reactions using molecularoxygen and NADPH and play an important role in thebiosynthesis of many primary and specialized meta-bolites. Such expansion of gene families creates a fertileground for the evolution of novel metabolic activities,either within the same enzyme or between duplicateenzymes, over short evolutionary time scales (Wenget al., 2012).

Irrespective of the mode of duplication, most dupli-cate genes are lost over time (Lynch and Conery, 2000;Moghe and Shiu, 2014). However, some may be selec-tively retained because of the acquisition of a new, bene-ficial function (neofunctionalization) or partitioning theancestral functions between duplicate partners (sub-functionalization), via coding sequence and/or regulatoryevolution (Prince and Pickett, 2002). In the case of dupli-cates of primary metabolism genes, while most of themmay be lost through time, some retained duplicates maypartially divert the flux from the primary metabolicpathway into catalysis of a new product, in a manner thatincreases plantfitness. Subsequent integration of the novelreaction with existing or derived metabolic networks inthe plant would lead to a successful recruitment of a pri-mary metabolic enzyme into specialized metabolism.

THE RECRUITMENT OF PRIMARY METABOLICENZYMES INTO SPECIALIZED METABOLISMOCCURS VIA MANY PATHS

The process of recruitment may occur via multiplepaths, such as changes in the transcriptional and allo-steric regulation of an enzyme, partitioning of an en-zyme’s promiscuous activities, and simple changes insubstrate specificities, or via more complex structuralchanges, such as changes in protein-protein interactionsand the evolution of protein folds (Fig. 1). Such re-cruitment is documented to have occurred multipletimes in the plant kingdom, giving rise to a variety ofspecialized metabolite classes, namely pyrrolizidine,tropane, acridone and benzoxazinoid alkaloids, gluco-sinolates, acylsugars, and terpenoids (Table I). Wediscuss the emergence of each of these classes in greaterdetail below, starting with the simplest cases of re-cruitment and moving to the more complex scenarios.

Evolution of Transcriptional Regulation

The initial trigger for the recruitment of a primarymetabolic enzyme into specialized metabolism mayoccur even before the gene that encodes the enzyme isexpressed. As seen in tobacco (Nicotiana tabacum), ex-pression divergence between duplicate genes may playan important role in such recruitment. The tobaccoQUINOLATE PHOSPHORIBOSYLTRANSFERASE(QPT) is an enzyme that converts quinolate to nicotinateribonucleotide, which is a precursor for both NAD andnicotine biosynthesis (Sinclair et al., 2000). While NADplays an important role in numerous reactions in primaryand specialized metabolism, nicotine is a specializedmetabolite important for herbivory defense (Steppuhnet al., 2004). Tobacco has two copies of QPT, QPT1 andQPT2, which are 94% identical to each other but havedifferent expression profiles (Shoji andHashimoto, 2011).While QPT1 is constitutively expressed in leaves andflowers at a basal level,QPT2 is most highly expressed inthe root tissue and contributes most of the QPT tran-scripts in leaves and flowers. In addition, unlike QPT1,

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Enzyme Recruitment to Specialized Metabolism



Figure 1. Mechanisms that lead to the divergence of duplicate genes and their subsequent recruitment to specializedmetabolism. A,After duplication, the duplicate gene gains new cis-regulatory elements, creating a different expression profile compared with theprogenitor metabolic gene. Divergence in spatial, temporal, and environmental responsiveness may lead to the evolution of newmetabolic pathways in the plant. B, A primary metabolic enzyme with main and side activities. After duplication, one copy retainsand is optimized for the side activity. C, A primary metabolic enzyme, which produces only a single product, gets duplicated. Theduplicated, diverged enzyme, however, is multifunctional and may produce several different products from the same substrate. D, Asingle amino acid change in the active site of a duplicate enzyme disrupts interaction with a substrate or a cofactor, abolishing theoriginal activity and potentially leading to the gain of a new activity. E, The progenitor primary metabolic enzyme is allostericallyinhibited by the endproduct of the pathway. This property is lost in the specializedmetabolic enzyme due to aC-terminal deletion. Inthe absence of such regulation, the steady-state levels of some of the pathway intermediates may increase, leading to novel

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the expression ofQPT2 is inducible upon wounding andby methyl jasmonate treatment (Shoji and Hashimoto,2011). It was found that these two genes originated in aNicotiana ancestor very recently and subsequently un-derwent cis-regulatory divergence (Fig. 1A). QPT2 wasfound to have gained three binding sites for the ethylene

response factor ERF189, a transcription factor regulatinggenes involved in nicotine biosynthesis (Shoji et al., 2010).These binding sites can act in an additive or syner-gistic fashion to increase QPT2 expression (Shoji andHashimoto, 2011). It was postulated that the ability toproduce nicotine pathway intermediates rapidly upon

Figure 1. (Continued.)metabolites in the cell. F, As in the case of TSa, close association of the two enzyme subunits largely prevents the pathway inter-mediate from being accessible to other enzymes. However, duplication of a single subunit followed by divergence leads to a novel,previously unseen product entering the cellular metabolite pools. G, The three enzyme models shown are highly divergent at theprimary sequence level; however, they preserve the a/b-hydrolase fold, which allows them to maintain similar activities.

Table I. Examples of the recruitment of specialized metabolism enzymes from primary metabolism

Additional examples of potential recruitment events that occurred very early in angiosperm and plant phylogeny are noted by Weng (2014).

No. Primary Metabolic Enzyme PathwayRelated Specialized

Metabolic EnzymePathway

Plant Clades Where

Primarily

Found/Studied

Reference

1 Quinolatephosphoribosyltransferase1

NADbiosynthesis

Quinolatephosphoribosyltransferase2

Methyljasmonate-induciblenicotinebiosynthesis

Tobacco Shoji andHashimoto(2011)

2 Deoxyhypusine synthase Translationalelongation

Homospermidine synthase Pyrrolizidinealkaloidbiosynthesis

Asteraceae,Convolvulaceae

Reimannet al. (2004);Kalteneggeret al. (2013)

3 Cycloartenol synthase Steroidbiosynthesis

Lupeol synthase Lupeolbiosynthesis

Arabidopsis Herrera et al.(1998)


Triterpene synthase Triterpenoidsaponinbiosynthesis

Costus speciosus Kawano et al.(2002)


b-Amyrin synthase Avenacinbiosynthesis

Avena sativa Qi et al.(2004)

6 Spermidine synthase Spermidinebiosynthesis

Putrescine N-methyltransferase

Nicotinebiosynthesis

Solanaceae(Nicotiana spp.),Convolvulaceae

Biastoff et al.(2009)

7 Anthranilate synthase a2 Trpbiosynthesis

Anthranilate synthase a1 Acridonealkaloidbiosynthesis

Rutaceae(common rue[Ruta graveolens])

Bohlmannet al.(1996)

8 Isopropylmalate synthase Leubiosynthesis

IPMS3 Acylsugarbiosynthesis

Solanaceae(cultivated tomato[Solanumlycopersicum])

Ning et al.(2015)

9 Isopropylmalate synthase Leubiosynthesis

Methyl thioalkylmalatesynthase

Glucosinolatebiosynthesis

Brassicaceae Kroymannet al.(2001)

10 Trp synthase subunit a Trp biosynthesis IGL/BX1 Benzoxazinoidbiosynthesis

Gramineae(maize [Zea mays])

Frey et al.(1997, 2000)

11 Fatty acid-binding protein Fatty acidstorage

Chalcone isomerase Flavonoidbiosynthesis

Broadlydistributed

Ngaki et al.(2012)

12 Ser carboxypeptidase Proteinhydrolysis

Ser carboxypeptidase-likeacyltransferase

Acylationof diversemetabolites

Broadlydistributed

Milkowski andStrack (2004)

13 TPS-C/TPS-E terpenesynthase (ent-kaurenesynthase)

GAbiosynthesis

Terpene synthase Terpenoidbiosynthesis

Broadlydistributed

Trapp andCroteau(2001)

14 Carnitine acetyltransferase Fatty acidmodification

BAHD acyltransferases Acylationof diversemetabolites

Broadlydistributed

St-Pierre andDe Luca(2000)

15 b-Ketoacyl ACP synthase Fatty acidbiosynthesis

Chalcone synthase Flavonoidbiosynthesis

Broadlydistributed

Weng andNoel (2012)





wounding in multiple tissues, conferred by the diver-gence of QPT2, may have provided a fitness benefit tothe plant and ensured the retention of the duplicatecopies (Shoji and Hashimoto, 2011).

Expression divergence is a recurring theme in enzymerecruitment. In addition to expression divergence, theevolution of the enzymes themselves can contribute tofunctional diversification between duplicate copies. Suchevolution need not create an altogether new function, asseen in the case described below.

Evolution of Enzyme Promiscuity

Promiscuity is defined as the “coincidental catalysisof reactions other than the reaction(s) for which an en-zyme evolved” (Khersonsky and Tawfik, 2010; Fig. 1B).Metabolic enzymes may have varying degrees of pro-miscuity; for example, enzymes involved in specializedmetabolism have been proposed to be more promis-cuous compared with those involved in primary me-tabolism, which tend to be more restrictive in theirreaction space (Milo and Last, 2012; Weng and Noel,2012). However, despite being optimized over hun-dreds of millions of years of evolution, primary meta-bolic enzymes may still exhibit promiscuous activities,as evidenced by the emergence of pyrrolizidine alka-loids (PAs) in multiple plant lineages (Fig. 2A).

PAs are produced by plants in diverse dicot andmonocot families, such as theAsteraceae, Boraginaceae,Convolvulaceae, Fabaceae, and Orchidaceae (Reimannet al., 2004; Anke et al., 2008; Langel et al., 2010). Thefirst step in the pathway, catalyzed by the enzymeHOMOSPERMIDINE SYNTHASE (HSS), is the transferof an aminobutyl moiety from spermidine to putres-cine, producing homospermidine (Fig. 2A). Homo-spermidine is then converted by a series of downstreamreactions to different PAs, which play a role in defenseagainst herbivores (Hartmann, 1999). The HSS activityemerged independently in diverse plant families fromthe highly conserved and ubiquitously distributed en-zyme DEOXYHYPUSINE SYNTHASE (DHS), which isinvolved in protein hypusination in all eukaryoticspecies (Ober and Hartmann, 1999). When HSSs fromthese diverse species are compared with the DHSsfrom the same species, they are typically found to be60% to 90% identical to each other at the protein level(Reimann et al., 2004). Interestingly, DHS uses the sameaminobutyl donor as HSS (spermidine), but instead oftransferring the group to putrescine, it modifies a Lysresidue in the inactive form of the highly conservedprotein, EUKARYOTIC TRANSLATION INITIATIONFACTOR5A (eIF5A; Ober and Hartmann, 1999; Fig.2A). The deoxyhypusine residue product is then oxi-dized to hypusine by another enzyme. Hypusination ofeIF5A converts the protein to an active form, enabling itto take part in ribosomal translocation during transla-tional elongation (Saini et al., 2009).

Enzyme promiscuity has played an important role inthe emergence of the HSS activity. Biochemical studiesof DHS revealed that it can use both eIF5A and pu-trescine as acceptors, with eIF5A binding being themain activity both in vitro and in vivo (Ober andHartmann, 1999; Ober et al., 2003). In contrast, HSS canonly utilize putrescine and has lost the ability to bindeIF5A. Such transformation has occurred via positiveselection on certain amino acids in HSS, as shown inPA-producing species in the Convolvulaceae (Ober andKaltenegger, 2009; Kaltenegger et al., 2013). The di-vergence in activity between DHS and HSS was alsoaccompanied by a restriction in expression; in Aster-aceae species, DHS is broadly expressed while the

Figure 2. Reactionsof the primarymetabolic enzymes and their specializedmetabolic cousins in pyrrolizidine alkaloid, triterpenoid, and tropane alka-loid biosynthesis. Reactions forDHS/HSS (A), CAS/lupeol synthase/b-amyrinsynthase (B), and SPDS/PMT (C) are shown. The bond colors blue and redshow the most likely substrate from which the bond was derived.


Moghe and Last



specialized metabolic HSS activity is restricted pri-marily to roots (Ober and Hartmann, 1999).DHS/HSS recruitment is an example where the du-

plicate of a promiscuous enzyme was positively selectedand optimized for specializedmetabolism. In other cases,selection or drift may lead toward promiscuity, causing aduplicate of a specific enzyme to accept multiple sub-strates or produce multiple products. An example ofthis scenario is the sterol metabolism enzyme CYCLO-ARTENOL SYNTHASE (CAS), which, perhaps as a re-sult of its complex activity, has given rise to multiplemultifunctional specialized metabolic enzymes.CAS is conserved in all plants and is responsible for

producing essential sterols, including the membranecomponent ergosterol and brassinosteroid hormones(Phillips et al., 2006). CAS catalyzes a complex isom-erization reaction that involves the breaking of 11bonds and the formation of 11 new bonds, converting2,3-oxidosqualene to the stanol cycloartenol (Coreyet al., 1993; Fig. 2B). This primary metabolic enzyme isproposed to have been recruited into triterpenoid me-tabolite biosynthesis at least four times in the evolu-tionary history of plants: (1) as the primary metabolicenzyme lanosterol synthase in lanosterol biosynthesisin dicots (Suzuki et al., 2006); (2) as a multifunctionallupeol synthase identified in Arabidopsis (Herrera et al.,1998); (3) as a multifunctional triterpene synthase intriterpenoid saponin biosynthesis in the monocotC. speciosus (Kawano et al., 2002); and (4) as a b-amyrinsynthase in avenacin biosynthesis in the monocot oat(Avena strigosa; Qi et al., 2004; Fig. 2B). All of theseenzymes, including CAS, are collectively termedOXIDOSQUALENE CYCLASES (OSCs). Except forlanosterol synthase, each of these recruited enzymescyclizes oxidosqualene into nonsteroidal triterpenoidprecursors and, thus, diverts flux away from steroidbiosynthesis. Overall, OSCs belong to a large genefamily in plants, and several members of this family aremultifunctional enzymes, generating multiple prod-ucts such as lupeol, germanicol, and b-amyrin from2,3-oxidosqualene (Phillips et al., 2006; Fig. 1C). Phy-logenetic analysis suggests that CAS experienced anancestral duplication event, and this gave rise to alineage that itself experienced multiple duplications,producing multifunctional OSCs in different angio-sperm lineages (Phillips et al., 2006).

Divergence in Substrate Specificity

The evolution of promiscuity occurs via changes inenzyme sequence or structure. Such changes may alsooccur without the invocation of promiscuity. One ex-ample of this scenario is PUTRESCINE N-METHYLTRANSFERASE (PMT), which converts putrescine toN-methylputrescine, the committing step for nicotineand tropane alkaloid biosynthesis in plants in the orderSolanales (Fig. 2C). PMT has approximately 65% aminoacid identity to, and is evolutionarily derived from,the highly conserved primary metabolism enzyme

SPERMIDINE SYNTHASE (SPDS), which catalyzesthe conversion of putrescine to spermidine (Biastoffet al., 2009). While both enzymes use putrescine as asubstrate, theyhavedifferentdonors: PMTusesS-adenosyl-Met as a methyl donor, while SPDS employs decar-boxylated S-adenosyl-Met as an aminopropyl donor(Fig. 2C). Site-directed mutagenesis experiments sug-gest that a single amino acid change can convert theSPDS to PMT activity (Junker et al., 2013; Fig. 1D), al-though the actual evolutionary path taken by the du-plicate of SPDS is not known. The theme of restrictedexpression mentioned for HSS is seen here as well, withPMT expressed in the roots while SPDS is expressed tovarying extents in multiple plant tissues.

Changes in Allosteric Regulation

Amino acids and their pathway intermediates serveas precursors to diverse specialized metabolites, in-cluding some that are produced in relatively largequantities either constitutively or in response to envi-ronmental induction. Modifying the flux in amino acidbiosynthetic pathways, which may occur as a result ofenzyme duplication and divergence, could cause astrong decrease in plant fitness. Evolution appears tohave addressed this problem in at least three ways.First, as in the case of HSS, expression of the recruitedenzyme can be limited spatially or temporally so thatflux is diverted only in the tissue and at the time whenthe specialized metabolite is synthesized. Second, asdiscussed below, there are examples of committingenzymes that have lost allosteric regulation during re-cruitment (Fig. 1E). Such a loss of regulation, if coupledwith expression restriction, can potentially limit theimpact of recruitment on flux divergence. The thirdapproach, changing the subcellular localization of theduplicate enzyme, is discussed later in this review.

Amino acid biosynthetic pathways produce metabo-lites that feed into several primary and specializedmetabolic pathways. Hence, the carbon/nitrogen fluxthrough these pathways is tightly controlled atmultiplelevels of regulation. At the enzymatic level, regulation ofthe flux occurs via end-product feedback inhibition, andthis mechanism is documented for the production of di-verse amino acids. For example, ISOPROPYLMALATESYNTHASE (IPMS) is inhibited by Leu, Thr deaminaseby Ile, cystathionine g-synthase by S-adenosyl-Met, anddihydrodipicolinate synthase by Lys, to name a few(Curien et al., 2008). Another enzyme, ANTHRANILATESYNTHASE (AS), catalyzes the committing step of Trpbiosynthesis and is inhibited by micromolar concentra-tions of the amino acid end product (Li and Last, 1996).AS is made up of two subunits, ASa and ASb, and thereare two copies of ASa, namely ASa1 and ASa2, in thegenomes of Arabidopsis and common rue (Bohlmannet al., 1995, 1996). The ASa enzyme catalyzes the con-version of chorismate to anthranilate using ammonia as adonor (Fig. 3A; Bohlmann et al., 1995). Both ASa1 andASa2 are expressed in cell cultures of common rue, but





only ASa1 is highly induced upon fungal cell wall frag-ment elicitation (Bohlmann et al., 1995). Interestingly,ASa1 has lost the property of Trp feedback inhibition,perhaps due to amino acid substitutions in a well-conserved consensus element (Bohlmann et al., 1996).Thus, ASa1 induction results in the Trp concentration-independent production of anthranilate, which isconverted by the committing enzyme anthranilateN-methyltransferase to N-methylanthranilate, a pre-cursor for acridone alkaloid biosynthesis. Thus, the lossof feedback inhibition may provide a fitness benefit tothe plant by allowing the production of acridone alka-loids regardless of the levels of Trp in the cell.

A different mechanism for the recruitment of acommitting enzyme of amino acid biosynthesis via theloss of allosteric inhibition was recently discovered foracylsugar biosynthesis in glandular trichomes of culti-vated tomato (Solanum lycopersicum) and the wild to-mato Solanum pennellii (Ning et al., 2015). Acylsugarsare specialized metabolites produced in trichomes ofsolanaceous species, and tomato acylsucroses containaliphatic isoC5 produced from isovaleryl-CoA,which isderived from Leu biosynthesis (Schilmiller et al., 2010,2015; Ghosh et al., 2014). SlIPMS3, a trichome-expressedgene that encodes a C-terminally truncated variant ofIPMS, the committing enzyme of Leu biosynthesis, wasfound to map to a region of the cultivated tomato ge-nome that influences the accumulation of Leu-derivedacylsucroses. This truncation abolishes feedback inhi-bition by Leu without the loss of IPMS activity (Fig. 1E;Ning et al., 2015). The loss of allosteric regulation wasalso accompanied by a change in the regulation of geneexpression, with the new enzyme being specificallyexpressed in the apical cells of acylsugar-producinglong trichomes compared with the broad expressionof other tomato IPMS genes (Ning et al., 2015). Thus, asimple change in protein structure combined with anarrowing of expression to a specialized cell type al-lows the variant IPMS to funnel metabolic products toacylsugar biosynthesis.

Amore complicated set of changes in an IPMS-derivedfamily of enzymes is associated with Met-derived glu-cosinolate biosynthesis in species of the order Brassicales.Glucosinolates are hallmark metabolites of Brassicalesplants such asmustard (Brassica juncea), cabbage (Brassicaoleracea), and radish and impart a pungent taste and odorto these plants. These structurally diverse sulfur- andnitrogen-containing compounds are derived from carbonand amino acid metabolism, with Met-derived glucosi-nolates being predominant in Arabidopsis (Halkier andGershenzon, 2006). The large diversity in Met-derivedglucosinolates is made possible by the action of METHYL-THIOALKYLMALATE SYNTHASEs (MAMs), whichevolved from IPMS after the emergence of Brassicales.IPMS/MAMs in Arabidopsis are approximately 60%

Figure 3. Reactions of the primary metabolic enzymes and their spe-cialized metabolic cousins in acridone alkaloid, glucosinolate, andbenzoxazinoid biosynthesis. Reactions for ASa2/ASa1 (A), IPMS/MAM

(B), and TSa/IGL/BX1 (C) are shown. The bond colors blue and red showthe most likely substrate from which the bond was derived.


Moghe and Last



identical to each other (Benderoth et al., 2006), and it hasbeen shown that the MAM lineage experienced positiveselection after the two lineages separated (Benderothet al., 2006; deKraker andGershenzon, 2011). As seen forthe tomato SlIPMS3, loss of the C terminus of IPMS leadsto a Leu-insensitive enzymatic activity in the MAMs (deKraker and Gershenzon, 2011; Figs. 1E and 3B). How-ever, unlike SlIPMS3, which has a truncation caused bya simple point mutation, the C-terminal truncation ofMAMs is more complex, including reorganization of theIPMS exon-intron structure (Kroymann et al., 2001).Another difference is that MAM has a reduced Leubiosynthetic IPMS activity and an enhanced activity forsubstrates in glucosinolate biosynthesis. The MAM ac-tivity was optimized for glucosinolate biosynthesis notonly via the C-terminal deletion but also via other pointmutations in the coding sequence (Benderoth et al., 2006;de Kraker and Gershenzon, 2011). Despite these struc-tural and functional differences, the overall reaction ofthe two enzymes has remained similar: carrying out acondensation reaction of acetyl-CoAswith oxoacids (Fig.3B). Such similarity in reaction mechanisms between theprimary metabolic enzymes and their specialized me-tabolism cousins is another recurring theme amongrecruited enzymes (Figs. 2 and 3). It may reflectthe fact that the recruited enzymes, although experienc-ing a greater ability to explore the reaction space, are stilllimited by the constraints of active-site and overallprotein structures and the enzymes’ local metaboliteenvironments.

Extreme Reorganization of an Old Enzyme Complex

The evolution of the benzoxazinoid biosynthesispathway in maize and other grasses represents an evenmore complex case of primary metabolic enzyme re-cruitment, involving multiple rounds of gene duplica-tion, protein structure evolution, loss of protein-proteininteraction, and expression divergence. Benzoxazinoidssuch as 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-oneand its methylated derivative are specialized metabo-lites produced in many Gramineae species and somedicot plants, both constitutively and upon damage(Schullehner et al., 2008; Ahmad et al., 2011). This classof compounds originates from indole-3-glycerol phos-phate, an intermediate of Trp biosynthesis. Benzox-azinoids are produced in maize by two enzymes calledBENZOXAZINELESS1 (BX1) and INDOLE GLYC-EROL PHOSPHATE LYASE (IGL), which convertindole-3-glycerol phosphate to indole (Frey et al., 1997,2000, 2004; Fig. 3C). BX1 is developmentally regulatedin young seedlings, while IGL is induced upon her-bivory. The free indole produced by BX1 and IGL eithercan be used in the production of benzoxazinoids viathe action of downstream CYP enzymes or serve as avolatile signal for allelopathic and other multitrophiccommunications (Frey et al., 2000).The evolution of IGL/BX1 activities represents a

striking case study in protein evolution. The IGL/BX1

lineage originated from the a-subunit of the Trp syn-thase complex (TSa; Frey et al., 1997; Melanson et al.,1997) via a single gene duplication event prior to thedivergence between maize, wheat (Triticum aestivum),and barley (Hordeum vulgare) and then diversified intoIGL and BX1 activities via additional rounds of dupli-cation (Grün et al., 2005; Dutartre et al., 2012). Nor-mally, the TSa subunit is found in a complex with theTSb subunit, making a physical channel that passesindole, the product of TSa, over to TSb. TSb then con-verts the indole to Trp using Ser (Figs. 1F and 3C), andfree indole rarely leaves the TS complex. In addition,TSa cannot function in isolation in bacteria (Hyde et al.,1988) or Arabidopsis (Radwanski et al., 1995). In con-trast, the IGL and BX1 enzymes not only perform theTSa function without the TSb subunit, they do it moreefficiently than the active TSa subunit in the TS com-plex (Frey et al., 1997, 2000). It was postulated that suchdramatic increases in efficiencies occurred partly due toamino acid substitutions that increased the protrusionof a Glu residue further into the active site of TSa, po-tentially increasing the efficiency of indole-3-glycerolphosphate binding (Schullehner et al., 2008). Upon re-cruitment, BX1 maintained the subcellular localizationof TSa (chloroplast) but, in contrast to the housekeepingrole of TSa, underwent transcriptional reprogrammingto be expressed only in young seedlings (Frey et al.,1997).

In addition to several Gramineae species, which areall monocots, individual species in some dispersed di-cot families also produce benzoxazinoids (Dick et al.,2012). Such a distribution has probably occurred due toone or multiple independent TSa duplications in dicotsleading to an IGL activity (Schullehner et al., 2008). Thepaths taken by each of the duplicates toward recruit-ment may also be different. For example, an indolesynthase in Arabidopsis that evolved from TSa lacksthe chloroplast transit peptide of TSa and is localized inthe cytosol (Zhang et al., 2008). Taken together withIPMS, these observations suggest that some genes in-volved in primary metabolism may act as a fountain-head of novel specialized metabolic enzymes.

Conservation of Structural Folds amid Overall ProteinSequence Divergence

Most of the examples of recruitment described aboveare relatively recent. However, the evolution of somespecialized metabolic pathways has also been influ-enced by more ancient duplication events. The OSCgene family derived from CAS has experienced onesuch ancient recruitment into specialized metabolism,as discussed above (Phillips et al., 2006). Members ofOSC, and some other gene families, diverged exten-sively from each other and from their original progen-itors; however, they still maintain the overall proteinfolds important for catalysis. TERPENE SYNTHASES(TPSs) and SERINECARBOXYPEPTIDASE-LIKE (SCPL)enzymes are examples of this phenomenon (Fig. 1G).





TPSs

TPSs constitute one of the most diverse gene familiesof specialized metabolism. They play a role in the bio-synthesis of terpenes, with more than 70,000 productsdescribed so far (Vickers et al., 2014). TPSs facili-tate the cyclization of polyprenoid precursors suchas geranyl diphosphate, farnesyl diphosphate, andgeranylgeranyl diphosphate, partly by chaperoningtheir substrates in their active sites to achieve aproper orientation, and the degree of conformationalfreedom available for the substrate in the active sitemay influence the degree of promiscuity of the TPSs(Christianson, 2008).

In contrast to more phylogenetically restricted spe-cialized metabolites, terpenoids such as the hormoneGA, the steroid precursor squalene, and the carotenoidprecursor phytoene are important for plant survivaland are broadly conserved across all plants and evensome bacteria and fungi. An ancestral diterpene syn-thase, possibly related to those involved in the currentGA biosynthetic pathways, was postulated to havegiven rise to the subfamilies of TPSs involved in spe-cialized metabolism via a single recruitment event ap-proximately 300 million years ago (Trapp and Croteau,2001; Gao et al., 2012). Since their recruitment, TPSshave experienced intron gains, losses, and fusions at thegene level and multiple losses of a structural domain atthe protein level (Trapp and Croteau, 2001). They alsoexpanded into a large family composed of multiplesequence similarity-based subfamilies that show littleamino acid identity to each other and use awide varietyof substrates (Chen et al., 2011). TPSs can also be clas-sified into two classes based on their active-site struc-tures: class I enzymes have an a-helical bundle, andclass II enzymes contain a pair of double a-barrel folds(Christianson, 2008; Gao et al., 2012). The presence ofthese different domains causes enzymes of these twoclasses to catalyze different reactions: while class I en-zymes generate the carbocation intermediate requiredfor cyclization by heterolytic cleavage, class II enzymesuse protonation as a means to generate this intermedi-ate (Gao et al., 2012). The ancestral enzyme that gaverise to the two classes has been proposed to be a bi-functional enzyme with both folds, probably related toent-kaurene synthase, an enzyme in the GA biosyn-thetic pathway (Chen et al., 2011; Gao et al., 2012). Asubsequent gene duplication and domain partitioningis proposed to have given rise to the two structuralclasses of TPSs found today.

SCPL Enzymes

The emergence of the SCPL family of enzymes from aSERINE CARBOXYPEPTIDASE (SCP) is another ex-ample of ancestral recruitment (Milkowski and Strack,2004). SCPLs constitute a large gene family, with ap-proximately 51 members in the Arabidopsis genome(Fraser et al., 2007). Overall, SCPs and SCPLs are 30% to40% identical to each other (Milkowski and Strack,

2004). While carboxypeptidases perform protein cleav-age by hydrolyzing peptide bonds, SCPL enzymes havelost the ability to bind to peptides and instead catalyzea transesterification reaction between substrates.Despite the large differences in sequence identityand biochemical function, SCPLs share a commonprotein fold (the a/b-hydrolase fold) and a conservednonsuccessive Ser-His-Asp catalytic triad in the activesite with the SCPs, enabling their molecular mechanismto be very similar (Milkowski and Strack, 2004; Fig. 1G).While Ser carboxypeptidases use water for a nucleo-philic attack on the enzyme-peptide intermediate, theSCPLs use a variety of other metabolites, such as ma-late, coumarate, quinate, benzoate, and gallate, for theattack (Bontpart et al., 2015). The ability to use diversesubstrates makes SCPLs important for the biosynthesisof diverse specialized metabolites, including the cya-nogenic glycoside dhurrin in sorghum (Sorghum bicolor;Wajant et al., 1994), sinapoylmalate in Arabidopsis andother Brassicaceae species (Lehfeldt et al., 2000), an-thocyanins in carrot (Daucus carota; Edgar Gläßgen andUlrich Seitz, 1992), and avenacin biosynthesis in oat(Mugford et al., 2013).

CONCLUSION

The evolution of economically important and struc-turally diverse plant metabolic networks has capturedthe attention of researchers from various fields ofscience: from biology and chemistry to physics andcomputer science (Rios-Estepa and Lange, 2007;Khersonsky and Tawfik, 2010; Chae et al., 2014). Theadvent of comparative genomic approaches in the pastdecade has led to general insights into how the mas-sively diverse specialized metabolite diversity in plantshas evolved (DellaPenna and Last, 2008; Kroymann,2011; Weng et al., 2012). In this review, we focused onone mechanism, the recruitment of enzymes from pri-mary metabolism, that is critically important to theevolution of specialized metabolic pathways in plants(Table I).

The process of recruitment typically starts with agene duplication event. The newly created paralog(s)may undergo either subfunctionalization or neo-functionalization. Under the subfunctionalizationmodel, ancestral functions are partitioned among theduplicated genes, and both derived copies are essentialfor the complete ancestral function (Force et al., 1999).In neofunctionalization, one of the duplicate copies re-tains the ancestral function while the other experiencespositive selection to fix a beneficial novel function(Ohno, 1970). Most of the recruited enzymes describedabove can be inferred to have undergone neo-functionalization, via change in enzymatic activity(SPDS/PMT, IPMS/MAM, and CAS/lupeol/b-amyrinsynthase), through transcriptional divergence (QPT1/QPT2, ASa2/ASa1, IPMS/IPMS3, and TSa/IGL/BX1),and/or by the loss of allosteric regulation (ASa2/ASa1,IPMS/MAM, and IPMS/IPMS3). DHS/HSS, however,


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represents a special case of recruitment. Individually,the known models of duplicate gene evolution (i.e.subfunctionalization/neofunctionalization and escapefrom adaptive conflict) cannot explain the observeddiversification of HSS (Kaltenegger et al., 2013). DHS/HSS seems to have elements conforming to all threemodels of duplicate gene divergence. As more exam-ples of recruitment are discovered, it is likely that suchspecial cases will become more common, especially inthe context of promiscuous primary and specializedmetabolism enzymes.Irrespective of the explanatory model, functional di-

versification in all known cases has occurred primarilyin only one of the two copies, with the other paralogremaining highly similar to nonduplicated orthologs inother species. Diversification may occur at the level ofgene expression, enzyme activity, as well as networkand epistatic interactions (Jiang et al., 2011) of the du-plicate pair. Such diversification may be accompaniedby positive selection acting on one of the duplicates, asreported for MAM and HSS (Benderoth et al., 2006;Kaltenegger et al., 2013), and purifying selection on theother.After the duplication event, loss of one duplicate

gene is the most likely fate of newly duplicated genes(Lynch and Conery, 2000; Krokida et al., 2013), and theexamples noted above represent the rare survivingcases of successful retention and functional diversifi-cation. Although multiple factors were noted previ-ously to influence the retention of duplicate genes(Birchler and Veitia, 2012; Moghe et al., 2014), twoquestions regarding the duplicated enzyme may bequite relevant with regard to recruitment into special-ized metabolism. First, how rapidly can the enzymeactivity be optimized without causing a fitness disad-vantage to the plant (due to the diversion of flux fromthe primary pathway), and second, how rapidly can theenzyme integrate into existing biochemical networks orenable the creation of a new network? Insights may befound by considering characteristics of the enzymesand the reactions they catalyze.One relevant observation is that some primary met-

abolic enzymes, such as IPMS, TSa, DHS, and CAS,were repeatedly and independently recruited intospecialized metabolism in multiple plant lineages.What makes these enzymes susceptible to recruitment?It is clear that enzyme promiscuity and pliability areimportant factors driving metabolic evolution (Wengand Noel, 2012) and that some enzymes are morecapable of evolution (more evolvable) than others(Kirschner and Gerhart, 1998; O’Loughlin et al., 2006).Thus, the inherent ability of a duplicated enzyme toexplore a wider reaction space and generate productsthat integrate into existing metabolic networks mayinfluence whether it can be successfully recruited andretained. In addition to enzyme evolvability, the abilityof a substrate to be utilized by variant enzymes, theamenability of the reaction product to serve as a sub-strate for other enzymes, as well as the proximity of areaction product to metabolite hubs such as acyl-CoAs

(Weng et al., 2012) may also contribute to an enzymebeing a preferred target for recruitment. Perhaps pri-mary metabolic enzymes whose duplicates are morelikely to get integrated into existing cellular networksmay, in turn, experience selection for being moreevolvable (Earl and Deem, 2004).

Identifying the commonalities among the variousenzymes recruited into specialized metabolism canhelp inform a more exhaustive analysis of plantspecialized metabolic pathways. The properties ofrecruited enzymes, such as their sequence similarity toprimary metabolic relatives, restricted or altered do-mains of expression, similarity in reaction chemistries,and signatures of positive selection, can be used toidentify novel specialized metabolic enzymes. The factthat enzymes in many specialized metabolic biosyn-thetic pathways remain to be fully identified presentsan exciting opportunity for genetic, biochemical, andbioinformatic exploration of this area. Additional ex-amples of the types of recruitment mechanisms de-scribed in this review will almost certainly be revealedasmore specializedmetabolic networks are discovered.

ACKNOWLEDGMENTS

We thank members of the Solanum Trichome Project for helpful discussionsand Tony Schilmiller for comments on the article.

Received July 3, 2015; accepted August 13, 2015; published August 14, 2015.

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Something Old, Something New: Conserved Enzymes and the ... · Something Old, Something New: Conserved Enzymes and the Evolution of Novelty in Plant Specialized Metabolism1 Gaurav