Update on Metabolic Evolution

Something Old, Something New: Conserved Enzymes and the Evolution of Novelty in Plant Specialized 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 and ecological importance. This remarkable variety is a consequence of the diversity and rapid evolution of specialized metabolic pathways. These novel biosynthetic pathways originate via gene duplication or by functional divergence of existing genes, and they subsequently evolve through selection and/or drift. Studies over the past two decades revealed that diverse specialized metabolic pathways have resulted from the incorporation of primary metabolic enzymes. We discuss examples of enzyme recruitment from primary metabolism and the variety of paths taken by duplicated primary metabolic enzymes toward integration into specialized metabolism. These examples provide insight into processes by which plant specialized metabolic pathways evolve and suggest approaches to discover enzymes of previously uncharacterized metabolic networks.

The plant kingdom collectively produces hundreds of thousands of low molecular weight organic molecules traditionally known as secondary metabolites, some of which have been shown to play roles in abiotic and biotic stress responses (e.g. herbivory defense), beneficial insect interactions (e.g. pollinator attraction), and communication with other plant and nonplant species (e.g. allelopathy and legume-rhizobia interactions; Saito and Matsuda, 2010; Pichersky and Lewinsohn, 2011; Wink, 2011). These metabolites have been widely used throughout the course of human history as medicines, spices, perfumes, cosmetics, and pest-control agents as well as in religious and cultural rituals. For the past 150 years, there has been a strong focus on documenting the chemical diversity of secondary metabolites in the plant kingdom, leading to the discovery of diverse classes of compounds such as terpenes, flavonoids, alkaloids, phenylpropanoids, glucosinolates, and polyketides. These secondary compounds were historically differentiated from products of primary metabolism, such as sugars, amino acids, nucleic acids, and fatty acids, as being nonessential for plant survival (Sachs, 1874; Kossel, 1891; Hartmann, 2008). However, by the 1980s, important functional roles began to be elucidated for metabolites previously classified as secondary, such as the phenolics (e.g. plant-microbe interactions and UV-B light protection; Bolton et al., 1986; Peters et al., 1986; Li et al., 1993; Landry et al., 1995), alkaloids (defense against herbivory; Steppuhn et al., 2004), and terpenes

1 This work was supported by the National Science Foundation (grant nos. MCB–1119778 and IOS–1025636). * Address correspondence to [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.15.00994

(defense against herbivory, antimicrobial activities, and volatile pollinator attractants; Papadopoulou et al., 1999; Schiestl and Ayasse, 2001; Erbilgin et al., 2006; Nieuwenhuizen et al., 2009). This change in our understanding of their roles has led to the coining of a new term, specialized metabolites, for these compounds, both to acknowledge their importance and to reflect the fact that many of them are phylogenetically restricted (Pichersky et al., 2006; Pichersky and Lewinsohn, 2011). Although the structural diversity of specialized metabolites far exceeds that of primary metabolites, all specialized metabolite classes are ultimately derived from primary 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 alternatively from methylerythritol phosphate, which is derived from glycolytic pathway precursors (Kirby and Keasling, 2009). Nitrogen-containing alkaloids are derived from a variety of primary metabolites, including amino acids and purine nucleosides (Facchini, 2001). Over the past 20 years, an increasing number of specialized metabolic enzymes have also been found to have their origins in primary metabolic pathways (Weng, 2014). Such shifts in enzyme function are made possible primarily by the process of gene duplication, which is very common in plants. GENE DUPLICATION: THE DRIVER OF METABOLIC INNOVATION

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

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Enzyme Recruitment to Specialized Metabolism

sequences of single-copy genes, gene duplication and subsequent divergence, and biochemical noise (Weng et al., 2012). These processes may be further influenced by drift and/or selection acting on specific alleles and driving these alleles to fixation in populations. Gene duplication is a central genetic mechanism for generating novel specialized metabolic enzymes because, as long as the duplicated gene is not lost, it allows for the conservation of old functions while creating new opportunities for metabolic diversification. Gene duplication occurs in three ways, tandem, segmental, and whole-genome duplication, and the genes originating via these processes can diverge in expression and amino acid sequence, giving rise to new biochemical activities. Analysis of genome sequences in multiple plant species revealed that duplicates of primary metabolic enzyme genes arising out of tandem or whole-genome duplication have different evolutionary fates than those encoding duplicates of specialized metabolic enzymes. For example, genes that participate in primary metabolism, such as those involved in carbohydrate, lipid, amino acid, and nucleotide metabolism, generally tend to revert back to a single-copy status after tandem duplication, while specialized metabolic enzyme genes, such as those involved in sinapate ester and quercitin production in Arabidopsis (Arabidopsis thaliana) and kaempferol production in soybean (Glycine max), tend to be retained as duplicates (Chae et al., 2014). In contrast, after whole-genome duplication, both specialized and primary metabolic enzyme genes (e.g. those involved in glucosinolate and lignin biosynthesis and amino acid biosynthesis, respectively) tend to return to single-copy status (Chae et al., 2014). It has been proposed that the retention/loss biases between primary and specialized metabolism genes may occur because of a potential need for maintaining gene dosage among primary metabolic genes (Freeling and Thomas, 2006), although other features, such as network connectivity, expression level, breadth of expression, and degree of conservation, may also play an important role (Moghe et al., 2014). The retention of duplicates of genes involved in specialized metabolism is responsible for several specialized metabolic enzymes, such as terpene synthases (Falara et al., 2011), chalcone synthases/polyketide synthases (Durbin et al., 1995; Austin and Noel, 2003), and acyltransferases (D’Auria, 2006), being members of multigene gene families. An extreme example in this regard is the plant cytochrome P450 (CYP) gene family, which is estimated to include up to 1% of all genes in a variety of plant species (Mizutani and Ohta, 2010). CYP enzymes are primarily involved in catalyzing oxygenation and hydroxylation reactions using molecular oxygen and NADPH and play an important role in the biosynthesis of many primary and specialized metabolites. Such expansion of gene families creates a fertile ground for the evolution of novel metabolic activities, either within the same enzyme or between duplicate enzymes, over short evolutionary time scales (Weng et al., 2012). Plant Physiol. Vol. 169, 2015

Irrespective of the mode of duplication, most duplicate genes are lost over time (Lynch and Conery, 2000; Moghe and Shiu, 2014). However, some may be selectively retained because of the acquisition of a new, beneficial function (neofunctionalization) or partitioning the ancestral functions between duplicate partners (subfunctionalization), via coding sequence and/or regulatory evolution (Prince and Pickett, 2002). In the case of duplicates of primary metabolism genes, while most of them may be lost through time, some retained duplicates may partially divert the flux from the primary metabolic pathway into catalysis of a new product, in a manner that increases plant fitness. Subsequent integration of the novel reaction with existing or derived metabolic networks in the plant would lead to a successful recruitment of a primary metabolic enzyme into specialized metabolism.

THE RECRUITMENT OF PRIMARY METABOLIC ENZYMES INTO SPECIALIZED METABOLISM OCCURS VIA MANY PATHS

The process of recruitment may occur via multiple paths, such as changes in the transcriptional and allosteric regulation of an enzyme, partitioning of an enzyme’s promiscuous activities, and simple changes in substrate specificities, or via more complex structural changes, such as changes in protein-protein interactions and the evolution of protein folds (Fig. 1). Such recruitment is documented to have occurred multiple times in the plant kingdom, giving rise to a variety of specialized metabolite classes, namely pyrrolizidine, tropane, acridone and benzoxazinoid alkaloids, glucosinolates, acylsugars, and terpenoids (Table I). We discuss the emergence of each of these classes in greater detail below, starting with the simplest cases of recruitment and moving to the more complex scenarios. Evolution of Transcriptional Regulation

The initial trigger for the recruitment of a primary metabolic enzyme into specialized metabolism may occur even before the gene that encodes the enzyme is expressed. As seen in tobacco (Nicotiana tabacum), expression divergence between duplicate genes may play an important role in such recruitment. The tobacco QUINOLATE PHOSPHORIBOSYLTRANSFERASE (QPT) is an enzyme that converts quinolate to nicotinate ribonucleotide, which is a precursor for both NAD and nicotine biosynthesis (Sinclair et al., 2000). While NAD plays an important role in numerous reactions in primary and specialized metabolism, nicotine is a specialized metabolite important for herbivory defense (Steppuhn et al., 2004). Tobacco has two copies of QPT, QPT1 and QPT2, which are 94% identical to each other but have different expression profiles (Shoji and Hashimoto, 2011). While QPT1 is constitutively expressed in leaves and flowers at a basal level, QPT2 is most highly expressed in the root tissue and contributes most of the QPT transcripts in leaves and flowers. In addition, unlike QPT1, 1513

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Figure 1. Mechanisms that lead to the divergence of duplicate genes and their subsequent recruitment to specialized metabolism. A, After duplication, the duplicate gene gains new cis-regulatory elements, creating a different expression profile compared with the progenitor metabolic gene. Divergence in spatial, temporal, and environmental responsiveness may lead to the evolution of new metabolic pathways in the plant. B, A primary metabolic enzyme with main and side activities. After duplication, one copy retains and is optimized for the side activity. C, A primary metabolic enzyme, which produces only a single product, gets duplicated. The duplicated, diverged enzyme, however, is multifunctional and may produce several different products from the same substrate. D, A single amino acid change in the active site of a duplicate enzyme disrupts interaction with a substrate or a cofactor, abolishing the original activity and potentially leading to the gain of a new activity. E, The progenitor primary metabolic enzyme is allosterically inhibited by the end product of the pathway. This property is lost in the specialized metabolic enzyme due to a C-terminal deletion. In the absence of such regulation, the steady-state levels of some of the pathway intermediates may increase, leading to novel 1514

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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

Pathway

Related Specialized Metabolic Enzyme

1

Quinolate NAD phosphoribosyltransferase1 biosynthesis

2

Deoxyhypusine synthase

Translational elongation

3

Cycloartenol synthase

4

Cycloartenol synthase

Steroid biosynthesis Steroid biosynthesis

5

Cycloartenol synthase

6

Spermidine synthase

7

Anthranilate synthase a2

Trp biosynthesis

Anthranilate synthase a1

8

Isopropylmalate synthase

Leu biosynthesis

IPMS3

9

Isopropylmalate synthase

Leu biosynthesis

Methyl thioalkylmalate synthase

10

Trp synthase subunit a

Trp biosynthesis IGL/BX1

11

Fatty acid-binding protein

12

Ser carboxypeptidase

Fatty acid storage Protein hydrolysis

13

14

TPS-C/TPS-E terpene synthase (ent-kaurene synthase) Carnitine acetyltransferase

15

b-Ketoacyl ACP synthase

Steroid biosynthesis Spermidine biosynthesis

Pathway

Plant Clades Where Primarily Found/Studied

Quinolate Methyl Tobacco phosphoribosyltransferase2 jasmonateinducible nicotine biosynthesis Homospermidine synthase Pyrrolizidine Asteraceae, alkaloid Convolvulaceae biosynthesis Lupeol synthase Triterpene synthase

b-Amyrin synthase Putrescine N-methyl transferase

Chalcone isomerase Ser carboxypeptidase-like acyltransferase

GA biosynthesis

Terpene synthase

Fatty acid modification

BAHD acyltransferases

Fatty acid biosynthesis

Chalcone synthase

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

Lupeol biosynthesis Triterpenoid saponin biosynthesis Avenacin biosynthesis Nicotine biosynthesis

Arabidopsis Costus speciosus

Avena sativa

Solanaceae (Nicotiana spp.), Convolvulaceae Acridone Rutaceae alkaloid (common rue biosynthesis [Ruta graveolens]) Acylsugar Solanaceae biosynthesis (cultivated tomato [Solanum lycopersicum]) Glucosinolate Brassicaceae biosynthesis

Reference

Shoji and Hashimoto (2011)

Reimann et al. (2004); Kaltenegger et al. (2013) Herrera et al. (1998) Kawano et al. (2002) Qi et al. (2004) Biastoff et al. (2009) Bohlmann et al. (1996) Ning et al. (2015)

Kroymann et al. (2001) Benzoxazinoid Gramineae Frey et al. biosynthesis (maize [Zea mays]) (1997, 2000) Flavonoid Broadly Ngaki et al. biosynthesis distributed (2012) Acylation Broadly Milkowski and of diverse distributed Strack (2004) metabolites Terpenoid Broadly Trapp and biosynthesis distributed Croteau (2001) Acylation Broadly St-Pierre and of diverse distributed De Luca metabolites (2000) Flavonoid Broadly Weng and biosynthesis distributed Noel (2012)

response factor ERF189, a transcription factor regulating genes involved in nicotine biosynthesis (Shoji et al., 2010). These binding sites can act in an additive or synergistic fashion to increase QPT2 expression (Shoji and Hashimoto, 2011). It was postulated that the ability to produce 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 intermediate 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 the primary sequence level; however, they preserve the a/b-hydrolase fold, which allows them to maintain similar activities. Plant Physiol. Vol. 169, 2015

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Evolution of Enzyme Promiscuity

Figure 2. Reactions of the primary metabolic enzymes and their specialized metabolic cousins in pyrrolizidine alkaloid, triterpenoid, and tropane alkaloid biosynthesis. Reactions for DHS/HSS (A), CAS/lupeol synthase/b-amyrin synthase (B), and SPDS/PMT (C) are shown. The bond colors blue and red show the most likely substrate from which the bond was derived.

wounding in multiple tissues, conferred by the divergence of QPT2, may have provided a fitness benefit to the plant and ensured the retention of the duplicate copies (Shoji and Hashimoto, 2011). Expression divergence is a recurring theme in enzyme recruitment. In addition to expression divergence, the evolution of the enzymes themselves can contribute to functional diversification between duplicate copies. Such evolution need not create an altogether new function, as seen in the case described below. 1516

Promiscuity is defined as the “coincidental catalysis of reactions other than the reaction(s) for which an enzyme evolved” (Khersonsky and Tawfik, 2010; Fig. 1B). Metabolic enzymes may have varying degrees of promiscuity; for example, enzymes involved in specialized metabolism have been proposed to be more promiscuous compared with those involved in primary metabolism, which tend to be more restrictive in their reaction space (Milo and Last, 2012; Weng and Noel, 2012). However, despite being optimized over hundreds of millions of years of evolution, primary metabolic enzymes may still exhibit promiscuous activities, as evidenced by the emergence of pyrrolizidine alkaloids (PAs) in multiple plant lineages (Fig. 2A). PAs are produced by plants in diverse dicot and monocot families, such as the Asteraceae, Boraginaceae, Convolvulaceae, Fabaceae, and Orchidaceae (Reimann et al., 2004; Anke et al., 2008; Langel et al., 2010). The first step in the pathway, catalyzed by the enzyme HOMOSPERMIDINE SYNTHASE (HSS), is the transfer of an aminobutyl moiety from spermidine to putrescine, producing homospermidine (Fig. 2A). Homospermidine is then converted by a series of downstream reactions to different PAs, which play a role in defense against herbivores (Hartmann, 1999). The HSS activity emerged independently in diverse plant families from the highly conserved and ubiquitously distributed enzyme DEOXYHYPUSINE SYNTHASE (DHS), which is involved in protein hypusination in all eukaryotic species (Ober and Hartmann, 1999). When HSSs from these diverse species are compared with the DHSs from the same species, they are typically found to be 60% to 90% identical to each other at the protein level (Reimann et al., 2004). Interestingly, DHS uses the same aminobutyl donor as HSS (spermidine), but instead of transferring the group to putrescine, it modifies a Lys residue in the inactive form of the highly conserved protein, EUKARYOTIC TRANSLATION INITIATION FACTOR5A (eIF5A; Ober and Hartmann, 1999; Fig. 2A). The deoxyhypusine residue product is then oxidized to hypusine by another enzyme. Hypusination of eIF5A converts the protein to an active form, enabling it to take part in ribosomal translocation during translational elongation (Saini et al., 2009). Enzyme promiscuity has played an important role in the emergence of the HSS activity. Biochemical studies of DHS revealed that it can use both eIF5A and putrescine as acceptors, with eIF5A binding being the main activity both in vitro and in vivo (Ober and Hartmann, 1999; Ober et al., 2003). In contrast, HSS can only utilize putrescine and has lost the ability to bind eIF5A. Such transformation has occurred via positive selection on certain amino acids in HSS, as shown in PA-producing species in the Convolvulaceae (Ober and Kaltenegger, 2009; Kaltenegger et al., 2013). The divergence in activity between DHS and HSS was also accompanied by a restriction in expression; in Asteraceae species, DHS is broadly expressed while the Plant Physiol. Vol. 169, 2015

Enzyme Recruitment to Specialized Metabolism

specialized metabolic HSS activity is restricted primarily to roots (Ober and Hartmann, 1999). DHS/HSS recruitment is an example where the duplicate of a promiscuous enzyme was positively selected and optimized for specialized metabolism. In other cases, selection or drift may lead toward promiscuity, causing a duplicate of a specific enzyme to accept multiple substrates or produce multiple products. An example of this scenario is the sterol metabolism enzyme CYCLOARTENOL SYNTHASE (CAS), which, perhaps as a result of its complex activity, has given rise to multiple multifunctional specialized metabolic enzymes. CAS is conserved in all plants and is responsible for producing essential sterols, including the membrane component ergosterol and brassinosteroid hormones (Phillips et al., 2006). CAS catalyzes a complex isomerization reaction that involves the breaking of 11 bonds and the formation of 11 new bonds, converting 2,3-oxidosqualene to the stanol cycloartenol (Corey et al., 1993; Fig. 2B). This primary metabolic enzyme is proposed to have been recruited into triterpenoid metabolite biosynthesis at least four times in the evolutionary history of plants: (1) as the primary metabolic enzyme lanosterol synthase in lanosterol biosynthesis in dicots (Suzuki et al., 2006); (2) as a multifunctional lupeol synthase identified in Arabidopsis (Herrera et al., 1998); (3) as a multifunctional triterpene synthase in triterpenoid saponin biosynthesis in the monocot C. speciosus (Kawano et al., 2002); and (4) as a b-amyrin synthase in avenacin biosynthesis in the monocot oat (Avena strigosa; Qi et al., 2004; Fig. 2B). All of these enzymes, including CAS, are collectively termed OXIDOSQUALENE CYCLASES (OSCs). Except for lanosterol synthase, each of these recruited enzymes cyclizes oxidosqualene into nonsteroidal triterpenoid precursors and, thus, diverts flux away from steroid biosynthesis. Overall, OSCs belong to a large gene family in plants, and several members of this family are multifunctional enzymes, generating multiple products such as lupeol, germanicol, and b-amyrin from 2,3-oxidosqualene (Phillips et al., 2006; Fig. 1C). Phylogenetic analysis suggests that CAS experienced an ancestral duplication event, and this gave rise to a lineage that itself experienced multiple duplications, producing multifunctional OSCs in different angiosperm lineages (Phillips et al., 2006).

Divergence in Substrate Specificity

The evolution of promiscuity occurs via changes in enzyme sequence or structure. Such changes may also occur without the invocation of promiscuity. One example of this scenario is PUTRESCINE N-METHYL TRANSFERASE (PMT), which converts putrescine to N-methylputrescine, the committing step for nicotine and tropane alkaloid biosynthesis in plants in the order Solanales (Fig. 2C). PMT has approximately 65% amino acid identity to, and is evolutionarily derived from, the highly conserved primary metabolism enzyme Plant Physiol. Vol. 169, 2015

SPERMIDINE SYNTHASE (SPDS), which catalyzes the conversion of putrescine to spermidine (Biastoff et al., 2009). While both enzymes use putrescine as a substrate, they have different donors: PMT uses S-adenosylMet as a methyl donor, while SPDS employs decarboxylated S-adenosyl-Met as an aminopropyl donor (Fig. 2C). Site-directed mutagenesis experiments suggest that a single amino acid change can convert the SPDS to PMT activity (Junker et al., 2013; Fig. 1D), although the actual evolutionary path taken by the duplicate of SPDS is not known. The theme of restricted expression mentioned for HSS is seen here as well, with PMT expressed in the roots while SPDS is expressed to varying extents in multiple plant tissues.

Changes in Allosteric Regulation

Amino acids and their pathway intermediates serve as precursors to diverse specialized metabolites, including some that are produced in relatively large quantities either constitutively or in response to environmental induction. Modifying the flux in amino acid biosynthetic pathways, which may occur as a result of enzyme duplication and divergence, could cause a strong decrease in plant fitness. Evolution appears to have addressed this problem in at least three ways. First, as in the case of HSS, expression of the recruited enzyme can be limited spatially or temporally so that flux is diverted only in the tissue and at the time when the specialized metabolite is synthesized. Second, as discussed below, there are examples of committing enzymes that have lost allosteric regulation during recruitment (Fig. 1E). Such a loss of regulation, if coupled with expression restriction, can potentially limit the impact of recruitment on flux divergence. The third approach, changing the subcellular localization of the duplicate enzyme, is discussed later in this review. Amino acid biosynthetic pathways produce metabolites that feed into several primary and specialized metabolic pathways. Hence, the carbon/nitrogen flux through these pathways is tightly controlled at multiple levels of regulation. At the enzymatic level, regulation of the flux occurs via end-product feedback inhibition, and this mechanism is documented for the production of diverse amino acids. For example, ISOPROPYLMALATE SYNTHASE (IPMS) is inhibited by Leu, Thr deaminase by Ile, cystathionine g-synthase by S-adenosyl-Met, and dihydrodipicolinate synthase by Lys, to name a few (Curien et al., 2008). Another enzyme, ANTHRANILATE SYNTHASE (AS), catalyzes the committing step of Trp biosynthesis and is inhibited by micromolar concentrations of the amino acid end product (Li and Last, 1996). AS is made up of two subunits, ASa and ASb, and there are two copies of ASa, namely ASa1 and ASa2, in the genomes of Arabidopsis and common rue (Bohlmann et al., 1995, 1996). The ASa enzyme catalyzes the conversion of chorismate to anthranilate using ammonia as a donor (Fig. 3A; Bohlmann et al., 1995). Both ASa1 and ASa2 are expressed in cell cultures of common rue, but 1517

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only ASa1 is highly induced upon fungal cell wall fragment elicitation (Bohlmann et al., 1995). Interestingly, ASa1 has lost the property of Trp feedback inhibition, perhaps due to amino acid substitutions in a wellconserved consensus element (Bohlmann et al., 1996). Thus, ASa1 induction results in the Trp concentrationindependent production of anthranilate, which is converted by the committing enzyme anthranilate N-methyltransferase to N-methylanthranilate, a precursor for acridone alkaloid biosynthesis. Thus, the loss of feedback inhibition may provide a fitness benefit to the plant by allowing the production of acridone alkaloids regardless of the levels of Trp in the cell. A different mechanism for the recruitment of a committing enzyme of amino acid biosynthesis via the loss of allosteric inhibition was recently discovered for acylsugar biosynthesis in glandular trichomes of cultivated tomato (Solanum lycopersicum) and the wild tomato Solanum pennellii (Ning et al., 2015). Acylsugars are specialized metabolites produced in trichomes of solanaceous species, and tomato acylsucroses contain aliphatic isoC5 produced from isovaleryl-CoA, which is derived from Leu biosynthesis (Schilmiller et al., 2010, 2015; Ghosh et al., 2014). SlIPMS3, a trichome-expressed gene that encodes a C-terminally truncated variant of IPMS, the committing enzyme of Leu biosynthesis, was found to map to a region of the cultivated tomato genome that influences the accumulation of Leu-derived acylsucroses. This truncation abolishes feedback inhibition by Leu without the loss of IPMS activity (Fig. 1E; Ning et al., 2015). The loss of allosteric regulation was also accompanied by a change in the regulation of gene expression, with the new enzyme being specifically expressed in the apical cells of acylsugar-producing long trichomes compared with the broad expression of other tomato IPMS genes (Ning et al., 2015). Thus, a simple change in protein structure combined with a narrowing of expression to a specialized cell type allows the variant IPMS to funnel metabolic products to acylsugar biosynthesis. A more complicated set of changes in an IPMS-derived family of enzymes is associated with Met-derived glucosinolate biosynthesis in species of the order Brassicales. Glucosinolates are hallmark metabolites of Brassicales plants such as mustard (Brassica juncea), cabbage (Brassica oleracea), and radish and impart a pungent taste and odor to these plants. These structurally diverse sulfur- and nitrogen-containing compounds are derived from carbon and amino acid metabolism, with Met-derived glucosinolates being predominant in Arabidopsis (Halkier and Gershenzon, 2006). The large diversity in Met-derived glucosinolates is made possible by the action of METHYLTHIOALKYLMALATE SYNTHASEs (MAMs), which evolved from IPMS after the emergence of Brassicales. IPMS/MAMs in Arabidopsis are approximately 60% Figure 3. Reactions of the primary metabolic enzymes and their specialized metabolic cousins in acridone alkaloid, glucosinolate, and benzoxazinoid biosynthesis. Reactions for ASa2/ASa1 (A), IPMS/MAM

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

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identical to each other (Benderoth et al., 2006), and it has been shown that the MAM lineage experienced positive selection after the two lineages separated (Benderoth et al., 2006; de Kraker and Gershenzon, 2011). As seen for the tomato SlIPMS3, loss of the C terminus of IPMS leads to a Leu-insensitive enzymatic activity in the MAMs (de Kraker and Gershenzon, 2011; Figs. 1E and 3B). However, unlike SlIPMS3, which has a truncation caused by a simple point mutation, the C-terminal truncation of MAMs is more complex, including reorganization of the IPMS exon-intron structure (Kroymann et al., 2001). Another difference is that MAM has a reduced Leu biosynthetic IPMS activity and an enhanced activity for substrates in glucosinolate biosynthesis. The MAM activity was optimized for glucosinolate biosynthesis not only via the C-terminal deletion but also via other point mutations in the coding sequence (Benderoth et al., 2006; de Kraker and Gershenzon, 2011). Despite these structural and functional differences, the overall reaction of the two enzymes has remained similar: carrying out a condensation reaction of acetyl-CoAs with oxoacids (Fig. 3B). Such similarity in reaction mechanisms between the primary metabolic enzymes and their specialized metabolism cousins is another recurring theme among recruited enzymes (Figs. 2 and 3). It may reflect the fact that the recruited enzymes, although experiencing a greater ability to explore the reaction space, are still limited by the constraints of active-site and overall protein structures and the enzymes’ local metabolite environments.

Extreme Reorganization of an Old Enzyme Complex

The evolution of the benzoxazinoid biosynthesis pathway in maize and other grasses represents an even more complex case of primary metabolic enzyme recruitment, involving multiple rounds of gene duplication, protein structure evolution, loss of protein-protein interaction, and expression divergence. Benzoxazinoids such as 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one and its methylated derivative are specialized metabolites produced in many Gramineae species and some dicot plants, both constitutively and upon damage (Schullehner et al., 2008; Ahmad et al., 2011). This class of compounds originates from indole-3-glycerol phosphate, an intermediate of Trp biosynthesis. Benzoxazinoids are produced in maize by two enzymes called BENZOXAZINELESS1 (BX1) and INDOLE GLYCEROL PHOSPHATE LYASE (IGL), which convert indole-3-glycerol phosphate to indole (Frey et al., 1997, 2000, 2004; Fig. 3C). BX1 is developmentally regulated in young seedlings, while IGL is induced upon herbivory. The free indole produced by BX1 and IGL either can be used in the production of benzoxazinoids via the action of downstream CYP enzymes or serve as a volatile signal for allelopathic and other multitrophic communications (Frey et al., 2000). The evolution of IGL/BX1 activities represents a striking case study in protein evolution. The IGL/BX1 Plant Physiol. Vol. 169, 2015

lineage originated from the a-subunit of the Trp synthase complex (TSa; Frey et al., 1997; Melanson et al., 1997) via a single gene duplication event prior to the divergence between maize, wheat (Triticum aestivum), and barley (Hordeum vulgare) and then diversified into IGL and BX1 activities via additional rounds of duplication (Grün et al., 2005; Dutartre et al., 2012). Normally, the TSa subunit is found in a complex with the TSb subunit, making a physical channel that passes indole, the product of TSa, over to TSb. TSb then converts the indole to Trp using Ser (Figs. 1F and 3C), and free 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 contrast, the IGL and BX1 enzymes not only perform the TSa function without the TSb subunit, they do it more efficiently than the active TSa subunit in the TS complex (Frey et al., 1997, 2000). It was postulated that such dramatic increases in efficiencies occurred partly due to amino acid substitutions that increased the protrusion of a Glu residue further into the active site of TSa, potentially increasing the efficiency of indole-3-glycerol phosphate binding (Schullehner et al., 2008). Upon recruitment, BX1 maintained the subcellular localization of TSa (chloroplast) but, in contrast to the housekeeping role of TSa, underwent transcriptional reprogramming to be expressed only in young seedlings (Frey et al., 1997). In addition to several Gramineae species, which are all monocots, individual species in some dispersed dicot families also produce benzoxazinoids (Dick et al., 2012). Such a distribution has probably occurred due to one or multiple independent TSa duplications in dicots leading to an IGL activity (Schullehner et al., 2008). The paths taken by each of the duplicates toward recruitment may also be different. For example, an indole synthase in Arabidopsis that evolved from TSa lacks the chloroplast transit peptide of TSa and is localized in the cytosol (Zhang et al., 2008). Taken together with IPMS, these observations suggest that some genes involved in primary metabolism may act as a fountainhead of novel specialized metabolic enzymes.

Conservation of Structural Folds amid Overall Protein Sequence Divergence

Most of the examples of recruitment described above are relatively recent. However, the evolution of some specialized metabolic pathways has also been influenced by more ancient duplication events. The OSC gene family derived from CAS has experienced one such ancient recruitment into specialized metabolism, as discussed above (Phillips et al., 2006). Members of OSC, and some other gene families, diverged extensively from each other and from their original progenitors; however, they still maintain the overall protein folds important for catalysis. TERPENE SYNTHASES (TPSs) and SERINE CARBOXYPEPTIDASE-LIKE (SCPL) enzymes are examples of this phenomenon (Fig. 1G). 1519

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TPSs

TPSs constitute one of the most diverse gene families of specialized metabolism. They play a role in the biosynthesis of terpenes, with more than 70,000 products described so far (Vickers et al., 2014). TPSs facilitate the cyclization of polyprenoid precursors such as geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, partly by chaperoning their substrates in their active sites to achieve a proper orientation, and the degree of conformational freedom available for the substrate in the active site may influence the degree of promiscuity of the TPSs (Christianson, 2008). In contrast to more phylogenetically restricted specialized metabolites, terpenoids such as the hormone GA, the steroid precursor squalene, and the carotenoid precursor phytoene are important for plant survival and are broadly conserved across all plants and even some bacteria and fungi. An ancestral diterpene synthase, possibly related to those involved in the current GA biosynthetic pathways, was postulated to have given rise to the subfamilies of TPSs involved in specialized metabolism via a single recruitment event approximately 300 million years ago (Trapp and Croteau, 2001; Gao et al., 2012). Since their recruitment, TPSs have experienced intron gains, losses, and fusions at the gene level and multiple losses of a structural domain at the protein level (Trapp and Croteau, 2001). They also expanded into a large family composed of multiple sequence similarity-based subfamilies that show little amino acid identity to each other and use a wide variety of substrates (Chen et al., 2011). TPSs can also be classified into two classes based on their active-site structures: class I enzymes have an a-helical bundle, and class II enzymes contain a pair of double a-barrel folds (Christianson, 2008; Gao et al., 2012). The presence of these different domains causes enzymes of these two classes to catalyze different reactions: while class I enzymes generate the carbocation intermediate required for cyclization by heterolytic cleavage, class II enzymes use protonation as a means to generate this intermediate (Gao et al., 2012). The ancestral enzyme that gave rise to the two classes has been proposed to be a bifunctional enzyme with both folds, probably related to ent-kaurene synthase, an enzyme in the GA biosynthetic pathway (Chen et al., 2011; Gao et al., 2012). A subsequent gene duplication and domain partitioning is proposed to have given rise to the two structural classes of TPSs found today. SCPL Enzymes

The emergence of the SCPL family of enzymes from a SERINE CARBOXYPEPTIDASE (SCP) is another example of ancestral recruitment (Milkowski and Strack, 2004). SCPLs constitute a large gene family, with approximately 51 members in the Arabidopsis genome (Fraser et al., 2007). Overall, SCPs and SCPLs are 30% to 40% identical to each other (Milkowski and Strack, 1520

2004). While carboxypeptidases perform protein cleavage by hydrolyzing peptide bonds, SCPL enzymes have lost the ability to bind to peptides and instead catalyze a transesterification reaction between substrates. Despite the large differences in sequence identity and biochemical function, SCPLs share a common protein fold (the a/b-hydrolase fold) and a conserved nonsuccessive Ser-His-Asp catalytic triad in the active site with the SCPs, enabling their molecular mechanism to be very similar (Milkowski and Strack, 2004; Fig. 1G). While Ser carboxypeptidases use water for a nucleophilic attack on the enzyme-peptide intermediate, the SCPLs use a variety of other metabolites, such as malate, coumarate, quinate, benzoate, and gallate, for the attack (Bontpart et al., 2015). The ability to use diverse substrates makes SCPLs important for the biosynthesis of diverse specialized metabolites, including the cyanogenic glycoside dhurrin in sorghum (Sorghum bicolor; Wajant et al., 1994), sinapoylmalate in Arabidopsis and other Brassicaceae species (Lehfeldt et al., 2000), anthocyanins in carrot (Daucus carota; Edgar Gläßgen and Ulrich Seitz, 1992), and avenacin biosynthesis in oat (Mugford et al., 2013).

CONCLUSION

The evolution of economically important and structurally diverse plant metabolic networks has captured the attention of researchers from various fields of science: from biology and chemistry to physics and computer science (Rios-Estepa and Lange, 2007; Khersonsky and Tawfik, 2010; Chae et al., 2014). The advent of comparative genomic approaches in the past decade has led to general insights into how the massively diverse specialized metabolite diversity in plants has evolved (DellaPenna and Last, 2008; Kroymann, 2011; Weng et al., 2012). In this review, we focused on one mechanism, the recruitment of enzymes from primary metabolism, that is critically important to the evolution of specialized metabolic pathways in plants (Table I). The process of recruitment typically starts with a gene duplication event. The newly created paralog(s) may undergo either subfunctionalization or neofunctionalization. Under the subfunctionalization model, ancestral functions are partitioned among the duplicated genes, and both derived copies are essential for the complete ancestral function (Force et al., 1999). In neofunctionalization, one of the duplicate copies retains the ancestral function while the other experiences positive selection to fix a beneficial novel function (Ohno, 1970). Most of the recruited enzymes described above can be inferred to have undergone neofunctionalization, via change in enzymatic activity (SPDS/PMT, IPMS/MAM, and CAS/lupeol/b-amyrin synthase), 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, Plant Physiol. Vol. 169, 2015

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represents a special case of recruitment. Individually, the known models of duplicate gene evolution (i.e. subfunctionalization/neofunctionalization and escape from adaptive conflict) cannot explain the observed diversification of HSS (Kaltenegger et al., 2013). DHS/ HSS seems to have elements conforming to all three models of duplicate gene divergence. As more examples of recruitment are discovered, it is likely that such special cases will become more common, especially in the context of promiscuous primary and specialized metabolism enzymes. Irrespective of the explanatory model, functional diversification in all known cases has occurred primarily in only one of the two copies, with the other paralog remaining highly similar to nonduplicated orthologs in other species. Diversification may occur at the level of gene expression, enzyme activity, as well as network and epistatic interactions (Jiang et al., 2011) of the duplicate pair. Such diversification may be accompanied by positive selection acting on one of the duplicates, as reported for MAM and HSS (Benderoth et al., 2006; Kaltenegger et al., 2013), and purifying selection on the other. 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 the examples noted above represent the rare surviving cases of successful retention and functional diversification. Although multiple factors were noted previously to influence the retention of duplicate genes (Birchler and Veitia, 2012; Moghe et al., 2014), two questions regarding the duplicated enzyme may be quite relevant with regard to recruitment into specialized metabolism. First, how rapidly can the enzyme activity be optimized without causing a fitness disadvantage to the plant (due to the diversion of flux from the primary pathway), and second, how rapidly can the enzyme integrate into existing biochemical networks or enable the creation of a new network? Insights may be found by considering characteristics of the enzymes and the reactions they catalyze. One relevant observation is that some primary metabolic enzymes, such as IPMS, TSa, DHS, and CAS, were repeatedly and independently recruited into specialized metabolism in multiple plant lineages. What makes these enzymes susceptible to recruitment? It is clear that enzyme promiscuity and pliability are important factors driving metabolic evolution (Weng and Noel, 2012) and that some enzymes are more capable of evolution (more evolvable) than others (Kirschner and Gerhart, 1998; O’Loughlin et al., 2006). Thus, the inherent ability of a duplicated enzyme to explore a wider reaction space and generate products that integrate into existing metabolic networks may influence whether it can be successfully recruited and retained. In addition to enzyme evolvability, the ability of a substrate to be utilized by variant enzymes, the amenability of the reaction product to serve as a substrate for other enzymes, as well as the proximity of a reaction product to metabolite hubs such as acyl-CoAs Plant Physiol. Vol. 169, 2015

(Weng et al., 2012) may also contribute to an enzyme being a preferred target for recruitment. Perhaps primary metabolic enzymes whose duplicates are more likely to get integrated into existing cellular networks may, in turn, experience selection for being more evolvable (Earl and Deem, 2004). Identifying the commonalities among the various enzymes recruited into specialized metabolism can help inform a more exhaustive analysis of plant specialized metabolic pathways. The properties of recruited enzymes, such as their sequence similarity to primary metabolic relatives, restricted or altered domains of expression, similarity in reaction chemistries, and signatures of positive selection, can be used to identify novel specialized metabolic enzymes. The fact that enzymes in many specialized metabolic biosynthetic pathways remain to be fully identified presents an exciting opportunity for genetic, biochemical, and bioinformatic exploration of this area. Additional examples of the types of recruitment mechanisms described in this review will almost certainly be revealed as more specialized metabolic networks are discovered. ACKNOWLEDGMENTS We thank members of the Solanum Trichome Project for helpful discussions and 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 Evolution of Novelty in Plant Specialized Metabolism.

Plants produce hundreds of thousands of small molecules known as specialized metabolites, many of which are of economic and ecological importance. Thi...
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