Recent Progress in Deciphering the Biosynthesis of Aspartate-Derived Amino Acids in Plants

Molecular Plant

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Volume 3

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Number 1

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Pages 54–65

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January 2010

REVIEW ARTICLE

Recent Progress in Deciphering the Biosynthesis of Aspartate-Derived Amino Acids in Plants Georg Jander1 and Vijay Joshi Boyce Thompson Institute for Plant Research, 1 Tower Road, Ithaca, NY 14850, USA

ABSTRACT Plants are either directly or indirectly the source of most of the essential amino acids in animal diets. Four of these essential amino acids—methionine, threonine, isoleucine, and lysine—are all produced from aspartate via a well studied biosynthesis pathway. Given the nutritional interest in essential amino acids, the aspartate-derived amino acid pathway has been the subject of extensive research. Additionally, several pathway enzymes serve as targets for economically important herbicides, and some of the downstream products are biosynthetic precursors for other essential plant metabolites such as ethylene and S-adenosylmethionine. Recent and ongoing research on the aspartate-derived family of amino acids has identified new enzyme activities, regulatory mechanisms, and in vivo metabolic functions. Together, these discoveries will open up new possibilities for plant metabolic engineering. Key words:

Metabolic regulation; primary metabolism; Arabidopsis.

INTRODUCTION Unlike most plants, bacteria, and fungi, humans and other animals are unable to accomplish the de novo biosynthesis of all 20 protein amino acids. In particular, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine are considered essential amino acids because they must be obtained in animal diets or, in some cases, from other sources such as gut microflora in ruminant animals (Bach et al., 2005) or endosymbiotic bacteria in phloem-feeding insects (Douglas, 1998). Histidine is also sometimes classified as an essential amino acid for humans. Although it is required by young children, the essential nature of histidine for human adults is somewhat uncertain (Imura and Okada, 1998; WHO/FAO/UNU, 2007). Four of the essential amino acids (isoleucine, lysine, methionine, and threonine; Figure 1) are produced from aspartate in plants via a branched pathway (Figure 2) and are therefore commonly called the aspartate-derived amino acids. This pathway does not exist in mammals and, in some parts of the world, the availability of aspartate-derived amino acids in human and animal diets is sub-optimal. Because of this nutritional importance, aspartate-derived amino acid biosynthesis has been the subject of more extensive research than other plant amino acid biosynthesis pathways. Additional research interest in this pathway comes from the fact that it provides precursors for the biosynthesis of other essential plant metabolites, including S-adenosylmethionine and ethylene. The isoleucine biosynthesis pathway is also agriculturally relevant because acetolactate synthase, an enzyme in this pathway, is the target of several commercially important herbicides. It was recently demon-

strated that glycine can be synthesized from threonine in plants (Joshi et al., 2006). However, there are other pathways leading to glycine biosynthesis in plants, and it is also a metabolic intermediate in photorespiration. Therefore, glycine, which is also not essential in mammalian diets, is not generally classified among the aspartate-derived amino acids. Major field crops, which either directly or indirectly as animal feed make up the majority of the diets of most human populations, are deficient in one or more of the essential amino acids. These deficiencies include lysine and tryptophan in most grains (Debadov, 2003; Pfefferle et al., 2003), methionine and threonine in soybeans (Muntz et al., 1998), and methionine, cysteine, and isoleucine in potatoes (Stiller et al., 2007). Therefore, there has been an interest in manipulating the biosynthesis of essential amino acids in these crops to increase their nutritive value for humans and other animals. Improved understanding of plant amino acid biosynthesis pathways now makes it possible to engineer increased amino acid content using not only classical plant breeding, but also transgenic approaches. To date, the only commercially available transgenic plant with elevated amino acid content is high-lysine maize (Frizzi et al., 2008; Ufaz and Galili, 2008).

1 To whom correspondence should be addressed. E-mail [email protected], fax 607-254-1502, tel. 607-254-1365.

ª The Author 2009. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssp104, Advance Access publication 17 December 2009 Received 17 October 2009; accepted 13 November 2009

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Figure 1. Structures of the Four Aspartate-Derived Amino Acids.

However, the feasibility of increasing amino acid content has also been demonstrated with other species, such as elevated methionine in potatoes (Di et al., 2003), and elevated tryptophan in potatoes (Yamada et al., 2004), adzuki bean (Hanafy et al., 2007), and soybean (Inaba et al., 2007). Further research on biosynthesis of essential amino acids will undoubtedly produce new approaches that can be implemented to improve the nutritive value of food plants. Much is already known about the biosynthesis of essential amino acids in plants, and it is impossible to cover all relevant information within the scope of one article. Therefore, this review will provide an overview of the aspartate-derived amino acid pathway, with a particular focus on new discoveries that have been made in the past 5 years.

ASPARTATE KINASE, THE COMMITTING REACTION OF THE ASPARTATE-DERIVED AMINO ACID PATHWAY Aspartate kinase (EC 2.7.2.4), which catalyzes the formation of aspartyl-4-phosphate from aspartate, is the first reaction leading to the biosynthesis of methionine, threonine, isoleucine, and lysine. In the model plant Arabidopsis thaliana (Arabidopsis), five genes have been confirmed to encode aspartate kinase activity. Three of these are monofunctional aspartate kinases and two also encode homoserine dehydrogenase (EC 1.1.1.3), the first enzyme in the pathway leading to methionine, threonine, and isoleucine biosynthesis (Figure 2). As a group, the aspartate kinase enzymes are subject to complex allosteric regulation (Galili, 1995). This regulation, which involves not only downstream metabolites in the aspartatederived amino acid pathway, but also seemingly unrelated amino acids, suggests that aspartate kinase is an important checkpoint for balancing the relative flux of different plant amino acid biosynthesis pathways.

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Figure 2. Biosynthesis of Methionine, Threonine, Isoleucine, and Lysine via the Aspartate-Derived Amino Acid Biosynthesis Pathway in Plants. Known mechanisms of feedback inhibition (red lines) and activation (green lines) are indicated. AK, aspartate kinase; ALS, acetolactate synthase; ASDH, aspartate semialdahyde dehydrogenase; BCAT, branched chain amino acid aminotransferase; CBL, cystathionine beta-lyase; CGS, cystathionine gamma-synthase; DAPAT, diaminopimelate aminotransferase; DAPDC, diaminopimelate decarboxylase; DAPE, diaminopimelate epimerase; DHADH, dihydroxy-acid dehydratase; DHDPR, dihydrodipicolinate reductase; DHDPS, dihydrodipicolinate synthase; HSD, homoserine dehydrogenase; HSK, homoserine kinase; KARI, ketol-acid reductoisomerase; LKR, lysine ketoglutarate reductase; MGL, methionine gamma-lyase; MS, methionine synthase; SAMS, S-adenosylmethionine synthase; TA, threonine aldolase; TD, threonine deaminase; TS, threonine synthase.

The three monofunctional Arabidopsis aspartate kinases are all subject to feedback inhibition by lysine (Ghislain et al., 1994; Paris et al., 2002; Rognes et al., 2002), and one of the three enzymes is also synergistically inhibited by Sadenosylmethionine (Curien et al., 2007). Although regulation of aspartate kinase by lysine is also observed in microorganisms (Giovanelli et al., 1989; Rognes et al., 1980), regulation by S-adenosylmethionine has only been reported in plants. The two bifunctional aspartate kinase–homoserine dehydrogenase enzymes are subject to feedback inhibition by threonine and leucine. However, the two enzymes differ in their sensitivity to this allosteric inhibition (Curien et al., 2005), suggesting different roles in regulating flux through the pathway. Assays with cloned bifunctional aspartate kinases also show activation by several other amino acids, including alanine, cysteine, isoleucine, serine, and valine (Curien et al., 2005; Paris et al., 2003, 2002; Rognes et al., 2002). Given the physiological concentrations of amino acids that are found inside plant cells, alanine is likely to be the most important of these activators (Curien et al., 2005). The crystal structure of Arabidopsis monofunctional aspartate kinase with bound inhibitors suggests a mechanism

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by which allosteric regulation can occur (Mas-Droux et al., 2006a). Monofunctional plant aspartate kinases contain two regulatory ACT domains (Pfam 01842), which have also been found in other enzymes involved in amino acid metabolism. In the aspartate kinase crystal structure, lysine and S-adenosylmethionine are bound to one of the two ACT domains (Mas-Droux et al., 2006a). Structural modeling suggests that binding of lysine and S-adenosylmethionine will inhibit efficient binding of aspartate to the active site of the enzyme. Inhibition of aspartate kinase by exogenously added lysine can be lethal for plants, and selection for lysine resistance has identified mutations in the ACT binding domain (Heremans and Jacobs, 1995, 1997). As in the case of the monofunctional enzymes, activity of the bifunctional aspartate kinase–homoserine dehydrogenases is also regulated by means of ACT domains. The two enzymatic domains are separated by two ACT sites, one regulating aspartate kinase activity and the other regulating homoserine dehydrogenase activity. Binding of threonine to the aspartate kinase ACT site promotes binding of a second threonine to the ACT site that inhibits homoserine dehydrogenase activity (Paris et al., 2003). Aspartyl-4-phosphate, the product of the aspartate kinase reaction, is the substrate for aspartate semialdehyde dehydrogenase (EC 1.2.1.11). In comparison to aspartate kinase, this enzyme has not received much attention from researchers. Aspartate semialdehyde dehydrogenase activity in maize is inhibited by methionine and less so by lysine and threonine (Gengenbach et al., 1978). Although the Arabidopsis aspartate semialdehyde dehydrogenase has been identified and cloned based on similarity to microbial enzymes (Paris et al., 2002), there are no reports of any allosteric regulation. Also, unlike in the case of aspartate kinase, mutations in aspartate semialdehyde dehydrogenase have not been found in selections for plants that are resistant to allosteric inhibitors.

METHIONINE METABOLISM L-Aspartate-4-semialdehyde, the last common intermediate in the aspartate-derived amino acid pathway, is a substrate for both dihydrodipicolinate synthase (EC 4.2.1.52), the committing enzyme for lysine biosynthesis, and homoserine dehydrogenase (EC 1.1.1.3), the committing enzyme for threonine, methionine, and isoleucine biosynthesis (Figure 2). Regulation of homoserine production by the latter enzyme has been described above because it is part of a bifunctional enzyme with aspartate kinase. Homoserine kinase (EC 2.7.1.39), which catalyzes the formation of O-phosphohomoserine from homoserine, is the next step in the pathway leading to the formation of methionine. Experiments with pea and radish suggest that this enzymatic activity is subject to regulation by threonine, isoleucine, valine, and S-adenosylmethionine (Baum et al., 1983; Thoen et al., 1978). However, other research with the cloned Arabidopsis enzyme did not show any allosteric regulation (Lee and Leustek, 1999). Moreover, overexpression of homoserine kinase in Arabidopsis did not result in the

accumulation of the downstream metabolites O-phosphohomoserine, threonine, or methionine (Lee et al., 2005). Similarly, expression of bacterial homoserine kinase in potatoes did not increase accumulation of downstream amino acids (Rinder et al., 2008). Therefore, this enzyme is likely not a rate-limiting step for either threonine or methionine biosynthesis. Like aspartate-4-semialdehyde, O-phosphohomoserine is a branch point metabolite in aspartate-derived amino acid biosynthesis in plants. Both threonine synthase (EC 4.2.3.1), the committing enzyme for threonine biosynthesis, and cystathionine c-synthase (EC 2.5.1.48), the committing enzyme for the methionine branch of the pathway, use Ophosphohomoserine as a substrate (Figure 2). In this respect, plants differ from bacteria, where homoserine rather than Ophosphohomoserine is the last common intermediate in the threonine and methionine biosynthesis pathways (Gophna et al., 2005). Given its position as the committing enzyme to methionine biosynthesis, cystathionine-c-synthase is an important regulatory point in the pathway. Unlike in the case of homoserine kinase, overexpression of cystathionine c-synthase in Arabidopsis causes a significant increase in the methionine accumulation (Kim et al., 2002), suggesting that this enzyme is a regulated or rate-limiting step in the pathway. When cystathionine c-synthase activity is reduced with an antisense construct in transgenic plants, there is a large increase in accumulation of the substrate, O-phosphohomoserine, and the plants have severe growth defects (Gakie`re et al., 2000a; Kim and Leustek, 2000). Several post-translational mechanisms regulate the activity of cystathionine c-synthase in plants. Selection for Arabidopsis mutants that are resistant to ethionine resulted in overproduction of methionine due to missense mutations in cystathionine c-synthase (Chiba et al., 1999; Inaba et al., 1994; Ominato et al., 2002). Further experiments identified an N-terminal domain of 11–13 amino acids that regulates mRNA stability and thereby cystathionine c-synthase activity (Chiba et al., 1999; Ominato et al., 2002; Suzuki et al., 2001). Interestingly, S-adenosylmethionine rather than methionine acts as an effector that binds to the identified domain and thereby regulates mRNA stability (Chiba et al., 2003; Onouchi et al., 2004). S-adenosylmethionine binding causes not only cystathionine c-synthase translational arrest (Lambein et al., 2003; Onouchi et al., 2005), but also mRNA decay (Haraguchi et al., 2008). Translational arrest in response to S-adenosylmethionine occurred even in a rabbit reticulocyte lysate system (Onouchi et al., 2008), showing that this regulatory process can occur completely in the absence of other plant factors. The importance of the cystathionine c-synthase regulatory domain was further demonstrated by transforming tobacco with a version of the gene that does not encode the N-terminus (Hacham et al., 2002). The large increase in methionine-derived volatiles in these transgenic plants demonstrated increased flux through the methionine biosynthesis pathway as a direct result of elevated cystathionine c-synthase activity.

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An additional post-translational regulation of cystathionine c-synthase comes through proteolytic cleavage of the Nterminal regulatory domain (Loizeau et al., 2007). In the crystal structure of tobacco cystathionine c-synthase, the N-terminus is outside of the main body of the protein, suggesting that it might be accessible to proteolytic cleavage (Steegborn et al., 1999). Proteolytic removal of the regulatory domain increased enzyme activity and methionine accumulation (Loizeau et al., 2007). Similar regulation of cystathionine c-synthase activity may occur by a different mechanism in Arabidopsis, where a transcript with a 90-nucleotide internal deletion that removes the N-terminal regulatory domain has been identified in vivo (Hacham et al., 2006). This internally deleted cystathionine c-synthase has elevated enzyme activity relative to the complete protein. The activity of cystathionine b-lyase (EC 4.4.1.8), the next enzyme in the methionine biosynthesis pathway, has not been investigated very thoroughly. As in the case of homoserine kinase, this is likely because cystathionine b-lyase is subject to less extensive regulation than the branch point enzymes in the pathway. Overexpression of cystathionine b-lyase in Arabidopsis did not significantly affect methionine accumulation (Gakie`re et al., 2000b), suggesting that this enzyme is not a rate-limiting step. However, some regulation of cystathionine b-lyase at the level of transcription or translation does occur in Arabidopsis. For instance, if expression of cystathionine c-synthase is reduced, there is a small but significant increase in the abundance of cystathionine b-lyase (Gakie`re et al., 2000a). The final reaction in the methionine biosynthesis pathway is catalyzed by methionine synthase (EC 2.1.1.14). Both cytosolic and plastidic forms of methionine synthase have been identified in Arabidopsis (Ravanel et al., 2004). Whereas plastidic methionine synthase likely functions in de novo methionine biosynthesis, the cytosolic enzymes more likely function to regenerate methionine as part of the S-adenosylmethionine cycle (Figure 3A). The great majority of methionine in plants is not utilized for protein biosynthesis, but is instead converted to S-adenosylmethionine by S-adenosylmethionine synthase (EC 2.5.1.6). In turn, S-adenosylmethionine serves as an essential cofactor in numerous methylation reactions (Cantoni, 1975; Lu, 2000), and is also a precursor for the biosynthesis of ethylene and polyamines. S-adenosylhomocysteine that remains after methylation reactions is recycled back to methionine by the sequential activity of adenosylhomocysteinase and methionine synthase (Figure 3A). Given that most metabolic flux through methionine is directed toward S-adenosylmethionine, it is perhaps not surprising that reducing S-adenosylmethionine synthase activity increase methionine accumulation in plants. Knockout mutations in one of the four Arabidopsis S-adenosylmethionine synthases greatly increased the free methionine content of the plants (Goto et al., 2002; Shen et al., 2002). However, these plants show severe growth defects, most likely due to reduced availability of S-adenosylmethionine for essential plant methylation reactions.

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Figure 3. Plant Methylation Cycles Involving Methionine. (A) The S-adenosylmethionine cycle, which contributes to numerous methylation reactions. (B) The S-methylmethionine cycle, which contributes to phloem transport of sulfur to reproductive tissue. Solid lines indicate major metabolic flux; dashed lines indicate minor flux in this model. MS, methionine synthase; SAMS, S-adenosylmethionine synthase; SMM, S-methylmethionine; Met, methionine; MMT, methionine methyltransferase; HMT, homocysteine methyltransferase.

Lysine, or at least some metabolite derived from lysine, was recently shown to be an important regulator of S-adenosylmethionine synthase transcription and enzyme activity (Hacham et al., 2007). Inhibition of S-adenosylmethionine synthase causes reduced accumulation of S-adenosylmethionine. Since S-adenosylmethionine is also a regulator of cystathionine c-synthase, this permits indirect regulation of methionine biosynthesis by lysine. S-adenosylmethionine synthase activity in plants may also be regulated through S-nitrosylation of the enzyme (Lindermayr et al., 2005). This covalent attachment of NO to the cysteine residues can reduce S-adenosylmethionine synthase activity and thereby the availability of S-adenosylmethionine. An interesting, though as yet unproven, hypothesis is that since S-adenosylmethionine serves as a precursor for ethylene biosynthesis, S-nitrosylation may provide a regulatory link between NO and ethylene metabolism in plants. Synthesis and catabolism of methionine also occurs as part of the S-methylmethionine cycle. In this metabolic cycle, which is likely unique to plants, methionine and S-methylmethionine are inter-converted by the enzymes homocysteine methyltransferase (EC 2.1.1.10) and methionine methyltransferase (EC 2.1.1.12). S-methylmethionine is an abundant sulfur transport molecule in the phloem, and the S-methylmethionine cycle likely functions in phloem loading and unloading (Bourgis et al., 1999; Lee et al., 2008), as illustrated in Figure 3B. In this model, methionine in vegetative tissue is converted to S-methylmethionine, which can be transported to sink tissue such as seeds, where it is converted back to methionine.

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Although homocysteine methyltransferase is also found in other organisms, methionine methyltransferase has only been reported in plants (Ranocha et al., 2000). Knockout mutations of the single methionine methyltransferase genes in both maize and Arabidopsis show that this is a non-essential enzyme in plants (Kocsis et al., 2003; Tagmount et al., 2002). Mutations in HMT2, one of three Arabidopsis homocysteine methyltransferases, greatly increase seed methionine content but do not cause significant growth defects (Lee et al., 2008). Knockout mutations of the other two Arabidopsis homocysteine methyltransferases are lethal (Lee and Jander, unpublished observations), suggesting that they have an as yet unconfirmed but essential function in plant metabolism.

glycine biosynthesis from threonine in plants has also not yet been determined, and there are at least two other pathways to glycine biosynthesis (Figure 4). Possible functions include replenishment of glycine to the photorespiratory cycle and degradation of excess threonine to maintain metabolic homeostasis. Mathematical modeling of the aspartate-derived amino acid pathway suggests that threonine concentrations are quite sensitive to perturbation and play a central regulator role in the pathway (Curien et al., 2009). Nevertheless, Arabidopsis plants that are mutated in both threonine aldolases are viable as long as there is concomitant overexpression of threonine deaminase (Joshi et al., 2006).

THREONINE METABOLISM

ISOLEUCINE METABOLISM

Threonine synthase (EC 4.2.3.1) and cystathionine c-synthase compete to use O-phosphohomoserine as a substrate for threonine or methionine biosynthesis, respectively (Figure 2). Transgenic approaches to increase or decrease threonine synthase activity in Arabidopsis have the predicted effects on methionine accumulation. Overproduction of Escherichia coli threonine synthase caused growth defects that could be rescued by the addition of methionine (Lee et al., 2005). Arabidopsis threonine synthase point mutations (Bartlem et al., 2000) and antisense constructs in potatoes (Zeh et al., 2001) have been used to reduce threonine synthase activity and elevate methionine accumulation. Activity of threonine synthase in plants is up-regulated by S-adenosylmethionine, thereby providing further cross-talk between the methionine and threonine branches of the pathway (Amir et al., 2002). The crystal structure of threonine synthase, with and without S-adenosylmethionine, demonstrates a mechanism by which this allosteric regulation occurs (MasDroux et al., 2006b; Thomazeau et al., 2001). Specifically, conformational changes induced by the binding of S-adenosylmethionine bring the pyridoxal phosphate cofactor into the proper position relative to the active site of threonine synthase. Although adenosine monophosphate acts as an inhibitor of threonine synthase in vitro (Laber et al., 1999), physiological concentrations of adenosine monophosphate and modeling studies suggest that such regulation is unlikely to play a major role in vivo (Curien et al., 2003). Threonine aldolase (EC 4.1.2.5) is a threonine catabolic enzyme that catalyzes the formation of glycine and acetyldehyde. Mutations in one of two Arabidopsis threonine aldolases greatly increase the seed threonine content (Jander et al., 2004). Knockout mutations in the other Arabidopsis threonine aldolase produce albino seedlings and are lethal (Joshi et al., 2006). This lethal effect can be rescued by overexpression of threonine deaminase (EC 4.3.1.19), suggesting that excess threonine, rather than lack of glycine, causes the observed deleterious effects in Arabidopsis threonine aldolase mutants. However, the proximal cause of this threonine lethality has not yet been identified. The metabolic function of

2-Oxobutanoate, a metabolic precursor for isoleucine biosynthesis, can be produced from threonine by threonine deaminase (Mourad and King, 1995) or from methionine by methionine c-lyase (EC 4.4.1.11) (Goyer et al., 2007; Rebeille et al., 2006). The two enzymes contribute to a common pool of 2-oxobutanoate in Arabidopsis: methionine c-lyase transcription is down-regulated when threonine deaminase activity is increased with a feedback-insensitive mutation, and the isoleucine deficit in a threonine deaminase knockdown mutant is rescued by methionine gamma lyase overproduction (Joshi and Jander, 2009). Nevertheless, under normal growth conditions, threonine deaminase is likely to be the predominant route for isoleucine biosynthesis in Arabidopsis. Inhibition of threonine deaminase with the herbicide 2-(1cyclohexen-3(R)-yl)-S-glycine has lethal effects in Arabidopsis that can be rescued by addition of 2-oxobutanoate (Mourad and King, 1995; Szamosi et al., 1994). This suggests that methionine c-lyase cannot compensate completely for a lack of threonine deaminase activity, though another possibility is that methionine c-lyase activity is also inhibited by the herbicide. Transcription of methionine c-lyase is significantly upregulated by both salt and drought stress (Less and Galili, 2008). Increased enzyme activity under these conditions was

Figure 4. Pathways Leading to Glycine Formation in Plants: From 3Phosphoglycerate as a Branch Off Glycolysis, from Glyoxylate during Photorespiration, and from Threonine via Threonine Aldolase as a Branch Off the Aspartate-Derived Amino Acid Biosynthesis Pathway.

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verified by isotope labeling experiments that show incorporation of carbon from methionine into isoleucine (Joshi and Jander, 2009). Therefore, methionine c-lyase likely contributes to the large increase in isoleucine content that is observed in Arabidopsis and other plants in response to osmotic stress (Nambara et al., 1998). However, although elevated threonine deaminase activity increased isoleucine accumulation and root growth on high-salt medium, methionine c-lyase mutations in Arabidopsis did not result in increased sensitivity to osmotic stress (Joshi and Jander, 2009). Threonine deaminase in Arabidopsis is subject to allosteric regulation by isoleucine, leucine, and valine. Selection for Arabidopsis lines resistant to O-methylthreonine identified mutants with threonine deaminase that is insensitive to feedback inhibition, and greatly increased isoleucine content (Mourad and King, 1995). Site-directed mutagenesis of Arabidopsis threonine deaminase has identified two distinct sites of allosteric regulation (Garcia and Mourad, 2004; Wessel et al., 2000). Whereas threonine deaminase activity is decreased through conformational changes that are induced by leucine and isoleucine binding, enzymatic activity is increased by interaction with valine. Binding of branched-chain amino acids also influences the association and dissociation of threonine deaminase monomers. Whereas leucine and isoleucine promote the formation of less active dimers, addition of valine promotes the formation of more active threonine deaminase tetramers (Halgand et al., 2002). Acetolactate synthase (EC 2.2.1.6), which catalyzes the formation of 2-aceto-2-hydroxybutyrate from 2-oxobutanoate, consists of a large catalytic subunit and a smaller regulatory subunit. This enzyme not only functions in isoleucine biosynthesis from 2-oxobutanoate, but also catalyzes the first reaction in a parallel pathway leading from acetolactate to the other two branched-chain amino acids, valine and leucine (Coruzzi and Last, 2000). The large subunit by itself is already subject to allosteric regulation by branched-chain amino acids, and selection for valine-resistant Arabidopsis identified mutations in the gene encoding this enzyme (Wu et al., 1994). Nevertheless, this allosteric regulation by leucine, isoleucine, and valine is enhanced by addition of the small subunit (Lee and Duggleby, 2001). Two independent regulatory sites, one affecting inhibition by leucine and one inhibition by valine or isoleucine, have been identified (Lee and Duggleby, 2002). Acetolactate synthase in plants has been studied extensively because it is inhibited by sulfonylurea, triazolopyrimidine, imidazolinone, and pyrimidyl-oxo-benzoate herbicides (Haughn and Somerville, 1986, 1990; Jander et al., 2003; Mourad and King, 1992; Mourad et al., 1993; Sathasivan et al., 1990, 1991). Mutations that confer resistance to one or more of these herbicides have been found in numerous plant species, both in the laboratory and as the result of agricultural herbicide application in the field. Crystallization of the acetolactate synthase together with chlorsulfuron or imidazolinone herbicides shows that these do not bind to the active site, but rather block a channel that provides access to the

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active site of the enzyme (McCourt et al., 2006). Acetolactate synthase mutations that prevent binding of the herbicides to their target sites can provide resistance, but may also alter enzyme kinetics and binding of the cofactors flavin adenine dinucleotide, magnesium, and thiamine diphosphate (Chang and Duggleby, 1998; Mourad et al., 1995). The herbicide target sites in acetolactate synthase are also distinct from those that provide feedback inhibition by branched chain amino acids; mutant enzymes that are no longer subject to feedback regulation are nevertheless sensitive to herbicides (Wu et al., 1994). The next two enzymes in the isoleucine biosynthetic pathway, ketol-acid reductoisomerase (EC 1.1.1.86) and dihydroxyacid dehydratase (EC 4.2.1.9), remain largely uncharacterized in plants (Binder et al., 2007). Although Arabidopsis candidate genes have been identified based on similarities to microbial enzymes, their enzymatic activities and regulatory mechanisms have not been verified. As in the case of acetolactate synthase, these two enzymes also catalyze the corresponding steps in valine and leucine biosynthesis in microorganisms, and almost certainly also in plants. The final step in isoleucine, leucine, and valine biosynthesis is catalyzed by branched-chain amino acid transferase (EC 2.6.1.42). Complementation of yeast knockout mutations has verified this enzymatic activity for six Arabidopsis proteins (Diebold et al., 2002). These are targeted to different subcellular location in the plant, one in the mitochondria, two in the cytosol, and three in the plastids (Diebold et al., 2002). Branched-chain amino acid transferase also catalyzes the first step in the catabolism of these amino acids, and the varied sub-cellular localization of this enzyme may be necessary for the reaction to proceed in both directions within the confines of a single cell. In particular, the mitochondrial enzyme in Arabidopsis has been shown to function in the catabolism of all three branched-chain amino acids (Schuster and Binder, 2005).

LYSINE METABOLISM Dihydrodipicolinate synthase (EC 4.2.1.52), which competes with homoserine dehydrogenase for a common substrate, catalyzes the first reaction leading to lysine biosynthesis (Figure 2). In Arabidopsis, the two genes that encode dihydrodipicolinate synthases have been the subject of extensive research (Craciun et al., 2000; Sarrobert et al., 2000; Vauterin et al., 1999; Vauterin and Jacobs, 1994). In vivo substrate competition with homoserine dehydrogenase is indicated by the fact that knockout mutations of one of the two Arabidopsis dihydrodipicolinate synthases decrease lysine and increase threonine accumulation (Craciun et al., 2000; Sarrobert et al., 2000). However, overproduction of aspartate kinase, which increases overall flux into the aspartate-derived amino acid pathway, increases the accumulation of only threonine, but not lysine (Heremans and Jacobs, 1995). If feedbackinsensitive dihydrodipicolinate synthase instead of aspartate kinase is overproduced, there is a significant increase in the

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lysine content of the plants (Ben-Tzvi Tzchori et al., 1996; Falco et al., 1995; Karchi et al., 1994; Mazur et al., 1999). This suggests that as the committing enzyme of the pathway leading to lysine formation, dihydrodipicolinate synthase is subject to feedback regulation according to the lysine needs of the plant. Unlike dihydrodipicolinate synthases, other enzymes in the lysine biosynthesis pathway have received relatively little attention. Although it was long assumed that lysine biosynthesis in plants proceeds in a similar manner as in most bacteria (column A in Figure 5), recent research has identified key differences. The activity of three enzymes—dihydrodipicolinate reductase, diaminopimelate epimerase, and diaminopimelate decarboxylase—was confirmed based on in vitro assays and/or complementation of the corresponding E. coli mutations (Hudson et al., 2005). However, no genes with similarity to bacterial enzymes that convert tetrahydrodipicolinate into diaminopimelate (Figure 5) are found in the Arabidopsis genome. Instead, Arabidopsis uses a novel diaminopimelate aminotransferase enzyme (EC 2.6.1.83) (Hudson et al., 2006) for lysine biosynthesis. Moreover, the identification of a Synechocystis sp. functional ortholog of Arabidopsis diaminopimelate aminotransferases suggests that both higher plants and cyanobacteria produce lysinevia thepathwaythatisdistinctfrom those that are found in most other bacteria (Figure 5). Lysine in yeast and other fungi is not aspartate-derived, and instead is synthesized from 2-diaminoadipate, making this an entirely different pathway from that found in plants (Velasco et al., 2002). A bifunctional enzyme catalyzes the first two steps in lysine catabolism: lysine ketoglutarate reductase (EC 1.5.1.8) and saccharopine dehydrogenase (EC 1.5.1.9). Knockout mutations

Figure 5. Comparison of Biosynthesis Pathways Leading from Tetrahydrodipicolinate to Lysine in Plants and Bacteria. (A) E.g. Escherichia coli. (B) E.g. Arabidopsis and cyanobacteria. (C) E.g. Bacillus sphaericus. Adapted from Hudson et al. (2005).

in this Arabidopsis enzyme cause increased lysine accumulation in the seeds (Zhu et al., 2001). Several mechanisms have been identified that permit complex regulation of this lysine degradative pathway. In addition to the bifunctional enzyme, both monofunctional lysine ketoglutarate reductase and saccharopine dehydrogenase can be produced from the same Arabidopsis gene: premature termination at a polyadenylation site in an intron produces a monofunctional lysine ketoglutarate reductase (Tang et al., 2002) and an alternate transcript produced from an internal promoter results in the synthesis of monofunctional saccharopine dehydrogenase (Tang et al., 2000). Alone, these two monofunctional enzymes have different catalytic properties, because interactions between the two subunits of the bifunctional enzyme affect their enzymatic activities (Zhu et al., 2002). Although expression of feedback-insensitive dihydrodipicolinate synthase can lead to increased lysine accumulation in plants (Ben-Tzvi Tzchori et al., 1996; Falco et al., 1995; Karchi et al., 1994; Mazur et al., 1999), this is generally associated with an increase in lysine catabolism that limits the maximum lysine concentrations that can be achieved. Simultaneous feedbackinsensitive dihydrodipicolinate synthase and knockdown of lysine ketoglutarate reductase–saccharopine dehydrogenase leads to synergistic increase in plant lysine content (Zhu and Galili, 2003). Although the very high lysine concentrations that are achieved with this approach can have a negative effect on plant growth, this can be alleviated by expressing constructs from seed-specific promoters. This approach has been used to greatly increase lysine accumulation in the seeds of Arabidopsis and maize (Frizzi et al., 2008; Zhu and Galili, 2004). Although expression of several hundred genes was altered in these experiments, induction of lysine accumulation has been associated with relatively few other metabolic changes, particularly limited to the tricarboxylic acid cycle in Arabidopsis seeds (Angelovici et al., 2009). At least two other pathways for lysine degradation likely exist in higher plants, but have not yet been studied very extensively. Lysine decarboxylase (EC 4.1.1.18) activity, which catalyzes the formation of cadaverine from lysine, has been reported in pea seedlings (Icekson et al., 1986). However, although there are two genes with similarity to lysine decarboxylases (At1g50575 and At5g26140) encoded in the Arabidopsis genome, their potential role in lysine catabolism has not yet been investigated. A possible lysine aminotransferase in Arabidopsis (At2g13810; ALD1) may represent yet another pathway for lysine catabolism. In this case, the product of the reaction would be either piperidine-2-carboxylate or piperidine-3-carboxylate. However, there is as yet no in vivo evidence for this degradative pathway in Arabidopsis or other plants.

FUTURE RESEARCH OUTLOOK Due to the extensive genetic tools that are available for studying Arabidopsis, much of the recent research on plant

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amino acid biosynthesis has involved this model plant. Knowledge gained from Arabidopsis will continue to enable plant breeding and genetic engineering efforts to increase the accumulation of essential amino acids. However, although it is likely that the basic framework of the aspartate-derived amino acid biosynthesis (Figure 2) will present in all plants, there will almost certainly be species-specific divergence from this common pathway. For instance, differences in the sub-cellular localization of key enzymes, transport of amino acids from source to sink tissues, and environmental conditions can affect amino acid accumulation. Therefore, by studying the specific aspects of amino acid biosynthesis and transport in individual crop species, it will be possible to improve the success rate of future metabolic engineering projects. Ongoing genome sequencing efforts and the development of new plant breeding and genetic engineering tools will enable future research with crop plants in a manner that is currently only possible with Arabidopsis. For instance, the use of zinc finger nucleases will allow not only site-directed mutagenesis of endogenous plant genes, but also the targeted insertion of transgenes or expression silencing constructs in plant genomes (Shukla et al., 2009; Townsend et al., 2009). Large genetic mapping populations and new high-throughput genotyping methods (Canaran et al., 2008) will make it possible to take advantage of natural variation in amino acid biosynthesis pathways in breeding projects to improve the nutritional content of food crops. The use of multiple mutations or transgenes will permit engineering of amino acid biosynthesis pathways in a more targeted manner. Overproduction or knockdown of single enzymes has often resulted in either deleterious effects or an absence of the desired changes in amino acid accumulation. In the case of lysine, large increases in free amino acid levels were only achieved through simultaneous up-regulation of biosynthesis and decrease in catabolism (Frizzi et al., 2008; Galili, 1995). Similar approaches could also be used to increase the accumulation of other essential amino acids. For instance, simultaneous overexpression of cystathionine gammasynthase and silencing of methionine gamma-lyase may permit methionine accumulation in excess of that which can be achieved with either approach alone. In this way, agronomically relevant increases in methionine may be achieved in target crops such as potato and soybean. Transcriptional regulation is also an important aspect of amino acid metabolism that requires further investigation. Currently, most known regulation of the pathway shown in Figure 2 occurs post-translationally via allosteric regulation of enzyme activities. However, there is also evidence for transcriptional regulation of the genes encoding these enzymes, both in the course of development and in response to exogenous stimuli. Although it has been proposed that amino acid degradative pathways are regulated more frequently at the level of transcription (Less and Galili, 2008), the transcription factors that affect amino acid catabolism in this manner have not yet been identified.

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New insights into amino acid biosynthesis will be obtained through detailed metabolomic, proteomic, and transcriptional analyses of plants with natural or induced variation in amino acid accumulation. These approaches will make it possible to decipher previously unknown regulatory pathways and the seemingly unrelated effects on plant metabolism that are often observed when amino acid biosynthesis is altered. Such future research, which almost certainly will be conducted not only with Arabidopsis, but also with more economically important crop species will lead to a more complete understanding of plant amino acid metabolism.

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