Tyrosine Ammonia-lyases

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

Review

Plant Phenylalanine/Tyrosine Ammonia-lyases Jaime Barros1,2,3 and Richard A. Dixon1,2,3,* Aromatic amino acid deaminases are key enzymes mediating carbon flux from primary to secondary metabolism in plants. Recent studies have uncovered a tyrosine ammonia-lyase that contributes to the typical characteristics of grass cell walls and contributes to about 50% of the total lignin synthesized by the plant. Grasses are currently preferred bioenergy feedstocks and lignin is the most important limiting factor in the conversion of plant biomass to liquid biofuels, as well as being an abundant renewable carbon source that can be industrially exploited. Further research on the structure, evolution, regulation, and biological function of functionally distinct ammonia-lyases has multiple implications for improving the economics of the agri-food and biofuel industries.

Plant Aromatic Amino Acid Ammonia-lyase Enzymes The plant aromatic amino acid-lyase family of enzymes includes dedicated or monofunctional L-phenylalanine (Phe) (see Glossary) ammonia-lyase [PAL; enzyme commission (EC) 4.3.1.24] and bifunctional Phe/L-tyrosine (Tyr) ammonia-lyase (PTAL, EC 4.3.1.25). L-Histidine (His) ammonia-lyase (HAL, EC 4.3.1.3) is closely related to PAL and PTAL but is absent from plants [1]. In animals, HAL catalyzes the nonoxidative elimination of ammonia from His yielding urocanic acid, an endogenous photoprotectant against UV irradiation and an activator of T cells that modulate the immune system [2]. In plants and fungi, a Tyr (monofunctional) ammonia-lyase TAL (EC 4.3.1.23) has not been identified to date; instead, PAL occurs widely and PTALs are mainly restricted to the monocot grass family Poaceae (Figure S1 in the supplemental information online).

Highlights It has been generally accepted that all plants synthesize lignin from the aromatic amino acid L-phenylalanine. Recent studies indicate that true grasses (Poaceae) are able to make up to nearly half of their lignin from L-tyrosine via the enzyme L-tyrosine ammonia-lyase. Understanding the interface between tyrosine and phenylpropanoid metabolism is of particular interest because grasses include the major food crops of the world (e.g., corn, wheat, rice) as well as ideal bioenergy feedstocks (e.g., switchgrass, Miscanthus, Sorghum), and lignin is a key limiting factor in the conversion of plant biomass to liquid biofuels and an abundant renewable carbon source that can be industrially exploited.

Among the 20 protein amino acids, Phe, Tyr, and L-tryptophan include a phenyl ring in their chemical structure. All aromatic amino acids are essential in humans, meaning that the body cannot synthesize them and thus these amino acids must be obtained from the diet. Animals have lost these costly metabolic pathways and some herbicides and antibiotics take advantage of this fact, by inhibiting enzymes involved in aromatic amino acid biosynthesis and thereby being toxic to plants and microbes but not animals [3]. The aromatic amino acids are used for the synthesis of proteins in all organisms, but in plants also serve as precursors of numerous specialized metabolites, particularly phenylpropanoids (Figure 1) and alkaloids. These compounds function as pigments, substituted waxes, phytoalexins, hormones, and cell wall components [4]. Phe and Tyr are formed via the shikimate pathway from arogenate. This compound is converted into Phe by dehydration or into Tyr by dehydrogenation [5]. The first committed step in the phenylpropanoid pathway (Figure 1) is the deamination of Phe into cinnamate by PAL; subsequent formation of coumarate involves direct hydroxylation of the aromatic ring by trans-cinnamate 4-hydroxylase (C4H). Coumarate is first transformed into an activated CoA-ester, which is subsequently hydroxylated and esterified, resulting in the caffeate derivatives [6]. The biosynthesis of ferulate and sinapate is currently considered to proceed via aldehydes derived from reduction of the Coenzyme A esters in Arabidopsis (Arabidopsis thaliana) [7]. However, it has recently been shown that grasses of the Poaceae family use a pathway leading to the free phenolic acids more efficiently than dicots. This pathway generates coumarate directly from Tyr by PTAL, caffeate from coumarate by bifunctional coumarate 3-hydroxylase/ascorbate peroxidase (C3H/APX), and ferulate from caffeate by caffeic acid 3-O-methyltransferase (COMT) [8,9] (Figure 1). The so-called hydroxycinnamates are the building blocks for all phenylpropanoids. Cinnamate is a precursor for B-ring-deoxy-flavonoids and stilbenes, and a large portion of the volatile benzoate derivatives. Coumarate is a precursor for coumarins, and most flavonoids, including anthocyanins and isoflavones. Caffeate, ferulate, and sinapate are precursors for most lignins, lignans, and phenolic esters, and are also associated with waxes in suberin and cutin [10–12].

Trends in Plant Science, --, Vol. --, No. --

1BioDiscovery Institute, University of North Texas, Denton, TX 76203, USA 2Department of Biological Sciences, University of North Texas, Denton, TX 76203, USA 3Center for Bioenergy Innovation (CBI), Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

*Correspondence: [email protected]

https://doi.org/10.1016/j.tplants.2019.09.011 ª 2019 Elsevier Ltd. All rights reserved.

1

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

Sunlight + CO2

O2

Glossary

Photosynthesis Indole alkaloids Indole glucosinolates Serotonin Camalexin Auxin

Glucose Pentose phosphate pathway Tryptophan Shikimate pathway

Cyanogenic glucosides Tocopherol (vitamin E) Plastoquinones Betalains Isoquinoline alkaloids

Chorismate Arogenate Phenylalanine Pheny PAL

Tyrosine

Cinnamate

PTAL

C4H

Flavonoids Coumarins H-Lignin

4CL Coumarate Coumaroyl-CoA HCT Coumaroyl shikimate C3H/APX C3'H Caffeoyl shikimate CSE Caffeate C/G/S-Lignins COMT Lignans

Phenolic esters Phenolic waxes

Glucosinolates Xanthones Phenolamides

B-ring-deoxy-flavonoids B-ring-deoxy-s lbenes Benzoate deriva ves Salicylate

Ferulate

Sinapate Trends in Plant Science

Figure 1. Roles of Aromatic Amino Acids in Plants. In contrast to animals, all plants preserve the costly metabolic pathways for the biosynthesis of the aromatic amino acids L-phenylalanine, L-tyrosine, and L-tryptophan, which are used for the synthesis of proteins and also serve as precursors of key natural products involved in cell wall formation, physiological regulation, defense, and signaling. The first committed step in the phenylpropanoid pathway is the deamination of L-phenylalanine into cinnamate by L-phenylalanine ammonia-lyase (PAL); subsequent formation of coumarate involves direct hydroxylation of the aromatic ring by trans-cinnamate 4-hydroxylase (C4H). Some plant families (Figure S1 in the supplemental information online) can also form coumarate directly from tyrosine by bifunctional L-phenylalanine/L-tyrosine ammonia-lyase (PTAL). Dashed arrows indicate multiple intermediate reactions. Aromatic amino acids are shown in bold orange letters and free phenolic acids in bold green letters. Major plant natural products derived from aromatic amino acids are shown in grey shaded squares. The enzymes involved in specific reactions are: C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; HCT, hydroxycinnamoyl CoA shikimate/ quinate hydroxycinnamoyl transferase; C3ʹH, p-coumaroyl quinate/shikimate 3ʹ-hydroxylase; CSE, caffeoyl shikimate esterase; C3H/APX, bifunctional coumarate 3-hydroxylase/ascorbate peroxidase; COMT, caffeic acid/ 5-hydroxyferulic acid 3/5-O-methyltransferase.

PTALs are restricted to the family Poaceae (or Gramineae, also known as true grasses) within the order Poales, and in lesser degree to some orders of dicot plants such as Fabales, Malvales, Asterales, Caryophyllales, ferns, and Solanales (Figure S1 in the supplemental information online). Similarly, PTAL from the yeast Rhodosporidium toruloides turns over efficiently both Phe and Tyr [13]. Thus, in all these species, PTAL-derived TAL activity can provide an alternative route to the phenylpropanoid precursor coumarate, bypassing the hydroxylation of cinnamate by the membrane-bound cytochrome P450 (CYP) C4H (Figure 1). The question as to how grasses use both Phe and Tyr to feed the phenylpropanoid pathway has been recently addressed in the model monocot Brachypodium (Brachypodium distachyon). Possession of PTAL is associated with the unique composition of grass cell walls (elevated proportions of syringyl (S)-rich lignins and cell wall-bound coumarates), and

2

Trends in Plant Science, --, Vol. --, No. --

ADH: enzyme arogenate dehydrogenase, involved in aromatic amino acid biosynthesis. Alkaloids: family of natural organic compounds with a heterocyclic nitrogen structure and a wide range of pharmacological effects. Arbuscular mycorrhiza: type of mycorrhiza in which the fungus penetrates the cortical cells of the roots of a vascular plant. C3H/APX: enzyme coumarate 3-hydroxylase/cytosolic ascorbate peroxidase (bifunctional). C4H: enzyme trans-cinnamate 4-hydroxylase, catalyzing the first ring hydroxylation step in phenylpropanoid biosynthesis. COMT: enzyme caffeic acid 3O-methyltransferase, catalyzing O-methylation of both caffeic acid and coniferaldehyde. CYPs: cytochrome P450s monooxygenases, heme-containing, membrane-associated mixed function oxygenases. DOPA: aromatic a-amino-acid 3,4-dihydroxy-L-phenylalanine, also known as levodopa or L-DOPA. E1cB-elimination reaction: type of two-step chemical reaction (ionization and deprotonation) in which an -OH or -OR group and an acidic hydrogen eliminate to form an additional bond. E1cB stands for elimination unimolecular conjugate base. E2-elimination reaction: chemical reaction similar to E1cB involving a single-step elimination mechanism. E2 stands for bimolecular elimination. F5H: enzyme ferulate/coniferaldehyde 5-hydroxylase, the first committed step in S lignin biosynthesis. Friedel–Crafts mechanism: type of electrophilic aromatic substitution reaction used to attach an acyl (acylation) or an alkyl (alkylation) group to a molecule. G-lignin: guaiacyl lignin is the polymerization product of coniferyl alcohol. HAL: enzyme L-histidine ammonia-lyase, involved in histidine degradation in mammals. His: L-histidine is an alpha-amino acid with imidazole side chain. Horizontal gene transfer (HGT): the movement of DNA between organisms other than via vertical

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

PTAL channels Tyr in vivo to generate nearly half of the lignin deposited [8]. Possession of PTAL is also associated with the operation of a pathway to guaiacyl-lignan (G-lignan) and S-lignin that works efficiently at the level of free hydroxycinnamic acids [9]. The formation of lignin from Tyr raises the possibility of additional pathways for modifying lignification in major crops. Engineering such pathways could significantly improve the economics of the agricultural, food, and biofuel industries. Here, we discuss current knowledge on the substrate selectivity, evolution, and putative differential regulation of plant ammonia-lyase enzymes and their functions in coordinating plant specialized metabolism. Our aim is to highlight open questions that may stimulate further research.

Structural Determinants of Substrate Selectivity and Catalysis Conversion of PAL enzymes to PTALs or TALs, potentially by gene editing, could provide an interesting approach for plant metabolic engineering. Understanding the structural basis of the substrate specificities of plant ammonia-lyases will be necessary to achieve this goal. Dedicated TAL enzymes with residual PAL activity have been identified only in the bacteria Rhodobacter sp. and Saccharothrix sp. [14,15]. Phenylpropanoids are common plant natural products but are largely absent in bacteria (see next section for a description of the specialized role of PAL and TAL in bacteria). Both monofunctional PALs and TALs are very specific for their natural substrates. For example, bacterial TALs have a PAL/TAL activity ratio lower than 0.01, whereas Arabidopsis and parsley (Petroselinum crispum) PALs have PAL/TAL activity ratios greater than 10 000 [16]. Conversely, PTALs have the ability to use both Tyr and Phe with similar efficiency. Previously reported PTALs in monocot plants and yeast showed PAL/TAL activity ratios between 0.2 and 6 [8,16–18]. PTALs also deaminate the a-amino acid levodopa (DOPA) to yield caffeate [8,18,19]. The mechanism by which PTALs are able to use multiple substrates is still not fully understood. Previous studies suggest that a single His residue in PTALs is responsible for forming a hydrogen bond with the hydroxyl group of Tyr and DOPA (Figure 2A). By mutating this His89 to Phe in Rhodobacter sphaeroides TAL, the selectivity of the enzyme switched to a PAL with 18 000-fold reduction in TAL activity and 220-fold increase in PAL activity. The opposite replacement (Phe144 to His) in Arabidopsis PAL yielded an 18-fold increase in TAL activity and 80-fold reduction of PAL activity [16]. Still, there is evidence to suggest that some other residues, such as those involved in the flexibility of the active site lid loops (Figure 2A,B), are required for substrate binding and may also contribute to catalysis of PTALs in grasses [18,20,21]. On the basis of current knowledge, an interesting approach would be to identify the residues that control the TAL activity of PTALs in grasses in order to develop gene editing strategies to convert PTALs into monofunctional PALs and vice versa. This should enable us to investigate with precision the biological and physiological functions of PTALs in plants. Although some heterotetrameric forms of PAL have been reported [22,23], most aromatic amino acid ammonia-lyases are homotetramers composed of four identical subunits forming a single active site containing the modified amino acid cofactor 3,5-dihydro-5-methylidene-4H-imidazol- 4-one (MIO) (Figure 2A,B). The enzyme-associated MIO is formed by autocatalytic condensation during peptide folding from a highly conserved alanine-serine-glycine (ASG) tripeptide motif. MIO is found in PALs, TALs, PTALs, HALs, and aminomutases, but not in other members of the L-amino acid-lyase family such as aspartate ammonia-lyases [16]. Phylogenetically, PALs and PTALs share 20–30% sequence identity to HALs and less than 20% identity to aspartate ammonia-lyases [24]. The mechanism of ammonia elimination is expected to be different among these enzymes as the side chain of aspartate is an acid, whereas in His it is an imidazole ring, and in both Phe and Tyr is a phenyl ring. The biochemistry behind the exact mechanism of ammonia elimination of P(T)ALs is still under debate, and alternative mechanisms have been proposed for the role of the MIO cofactor in catalyzing this reaction (Figure 2C). In the most accepted mechanism, called an E1cB-elimination reaction, MIO performs a nucleophilic attack on the amino group of the substrate. A general acid/base catalysis removes the b proton creating a carbanion intermediate which collapses, resulting in elimination of the a-amino group and formation of the double bond between the a and b carbons. In the presence of a strong base, a similar concerted E2-elimination reaction can occur in a one-step simultaneous process. Evidence for this mechanism has been obtained from crystallographic, molecular mechanic,

transmission from parent to offspring. Hydroxycinnamates: class of natural aromatic acids (or phenylpropanoids) with a C6–C3 skeleton. KFB: Kelch repeat F-box protein, role in protein post-translational modification through ubiquitination. Lignins: class of complex phenolic polymers found in the secondary cell walls of the vascular tissues in plants; the canonical lignin units are p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S). Mediator: multiprotein complex that functions as a transcriptional coregulator by interacting with transcription factors. MIO: cofactor 3,5-dihydro-5methylidene-4H-imidazol- 4-one involved in the PAL/PTAL reaction. NST3 (SND1): NAC secondary wall thickening promoting 3 (or secondary wall-associated NAC domain 1) transcription factor. PAL: enzyme L-phenylalanine ammonia-lyase, the first committed step in the phenylpropanoid pathway. Phe: abbreviation for aromatic amino acid L-phenylalanine. Phenylpropanoids:: broad family of organic compounds synthesized by plants from the amino acids L-phenylalanine or L-tyrosine, including among others hydroxycinnamates, lignins, coumarins, and flavonoids. PTAL: enzyme L-phenylalanine/Ltyrosine (bifunctional) ammonialyase. P(T)ALs has been used throughout the manuscript to refer both PALs and PTALs. S-rich lignin: syringyl lignin is the polymerization product of sinapyl alcohol. TAL: enzyme L-tyrosine ammonialyase. Tyr: aromatic amino acid L-tyrosine. TyrAT: enzyme tyrosine aminotransferase.

Trends in Plant Science, --, Vol. --, No. --

3

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

(A) PcPAL (1W27, blue) RsTAL (2O6Y, gold)

Outer lid-loop

H148F L405V

Inner lid-loop

Phosp

A276S MIO (Ac ve site)

D453E

I415L

L-Phenylalanine (PAL)

(B)

PALs

H148F

MIO

A276s

L405V-Loop-I415L

L-Tyrosine (TAL)

D453E Phosphor.

(C)

L-DOPA (LAL)

E1cB- or E2-elimina on

Friedel-Cra s

PTALs

PAL/TAL/LAL TAL

Phe: R1=R2=H Tyr: R1=R2=H Levodopa: R1=H, R2=OH

Trends in Plant Science

Figure 2. Structural and Biochemical Differences between PALs and PTALs. (A) Ribbon diagram of monomers of monofunctional PAL from parsley (Petroselinum crispum, Protein Data Bank 1W27, in blue, PcPAL) superimposed on bacterial monofunctional TAL from Rhodobacter sphaeroides (Protein Data Bank 2O6Y, in gold, RsTAL). Inset shows the active site and the residues involved in catalytic activity colored according to the corresponding subunit (A, B, C, D) of the PcPAL homotetramer. PAL/TAL/LAL, phenylalanine/tyrosine/levodopa ammonia-lyase activities found in grass PTALs [8]. (B) Partial sequence alignment of selected PAL/PTAL/TAL proteins. The amino acids [alanine-serineglycine (ASG)] converted to the cofactor 4-methylidene imidazol-5-one (MIO) group [16], the residues proposed to be key for substrate selectivity between TAl/PAL (H148F, A276S, L405V, I415L, D453E), and the putative phosphorylation site [30] are highlighted in green and yellow, respectively. (C) Potential mechanisms for PAL/TAL/LAL catalysis still under debate: Friedel–Crafts type electrophilic acylation, E1cB (via a carbocation intermediate) and E2 (concerted) reaction mechanisms. The scheme shows the bonds that are attacked during the two reaction types (see [95] for further details). Abbreviations: Ath, Arabidopsis thaliana; Bd, Brachypodium distachyon; Bo, Bambusa oldhamii; E1cB, elimination unimolecular conjugate base; E2, bimolecular elimination; PAL, L-phenylalanine ammonia-lyase; Pc, P. crispum; PTAL, bifunctional L-phenylalanine/L-tyrosine ammonia-lyase; Pv, Panicum virgatum, Rs, Rhodobacter sphaeroides; TAL, L-tyrosine ammonia-lyase; Zm, Zea mays.

and kinetic isotope studies [25–27]. An alternative Friedel–Crafts mechanism has also been proposed. In this case, MIO performs an electrophilic attack on the substrate’s aromatic ring, resulting in the loss of aromaticity and leading to a delocalized carbocation on the phenyl ring. As a consequence of this acylation reaction, the acidity of the substrate’s b proton is increased and this proton is then removed by a general acid/base catalysis that facilitates the electronic rearrangement, resulting in the elimination of ammonia and double-bond formation between the a and b carbons [28,29].

4

Trends in Plant Science, --, Vol. --, No. --

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

These different hypotheses are supported by two different crystal structures from different organisms: parsley PAL [30] for the Friedel–Crafts-like mechanism and R. toruloides PTAL [31] for the E1cB mechanism. It therefore appears that the PAL and TAL reactions may occur by different catalytic mechanisms [25]. Interestingly, recent results from the first crystal structure of a monocot PTAL from Sorghum bicolor suggest that the deamination of Phe involves a Friedel-Crafts-type attack at the aromatic ring, whereas the deamination of Tyr proceeds through the attack of the amino group initiated by a single step E2-reaction mechanism [18]. Based on the above, it is clear that additional structural and mutational work is necessary to facilitate strategies for engineering designer PTALs. The formation of protein–ligand complex crystals has been reported to be challenging in ammonia-lyases [18,19]. It would nevertheless be valuable to design optimized protein expression constructs [32] to explore the cocrystallization of grass PTALs with their multiple substrates (Phe, Tyr, and DOPA) to help elucidate the residues involved in catalysis and ammonia elimination.

Origin and Evolution Understanding the evolution of PAL and PTAL proteins can provide important insights in the processes that facilitated the colonization of land by plants (500 million years ago). This was a decisive step towards the development of terrestrial ecosystems. Ancient plants were confronted with major challenges, including UV radiation, desiccation, lack of structural support, and the need to protect against herbivores and pathogens. The emergence of specialized metabolic pathways, among them the ability to deaminate aromatic amino acids to assemble phenylpropanoid compounds, was crucial to cope with many of these stresses. The biosynthesis of monolignols and flavonoids was vital for reproductive biology and protection against UV light and microbes. The evolution of lignin biosynthesis brought physical rigidity to support tall growth so that plants could compete for light, as well as enabling vascular transport [33,34]. Although the initial physiological advantage of phenolic compounds is not clear, it has been suggested that they were originally used as internal signaling molecules [35], while after the emergence of more complex structures they evolved to serve for defense against microorganisms and UV radiation, prevent desiccation, and as support for the vasculature [36,11]. PAL enzymes have been characterized in the cyanobacteria Nostoc punctiforme, with a still unknown function [37]. In Streptomyces maritimus, PAL supplies cinnamate for the biosynthesis of the bacteriostatic agent enterocin [38], and in Photorhabdus luminescens for the production of an antibiotic stilbene [39]. In addition, monofunctional TAL enzymes have also been characterized in the bacterium Saccharothrix espanaensis, to produce caffeate for the antibiotic saccharomicin [15], and Rhodobacter capsulatus, involved in the synthesis of coumarate as the chromophore of their photoactive yellow protein photoreceptor [14]. In the fungi kingdom, PALs have been reported in Amanita, Aspergillus, Neurospora, and other basidiomycetes, and PTALs have been reported in Rhodosporidium, Rhodotorula, and Sporobolomyces, where they are thought to participate in the catabolism of Phe and Tyr as a source of carbon and nitrogen [23,40,41]. Current phylogenetic studies [42] suggest that ammonia-lyases emerged in bacteria with a role in antimicrobial defense, were transferred via horizontal gene transfer (HGT) to an early fungal lineage, and were further acquired by land plants via arbuscular mycorrhizal symbiosis (Figure 3). However, a direct HGT between bacteria and aquatic or land plants via an ancient symbiosis cannot be excluded (dashed line in Figure 3). What seems clear from currently available genomes is that aromatic ammonia-lyases have not been acquired by endosymbiotic gene transfer from chloroplastic cyanobacteria, as these enzymes are absent in both red and green algal lineages. It is striking how red and green algae deposit lignin or lignin-like polymers while lacking known PAL homologous genes [36,43,44]. It would be interesting to investigate further the genetic mechanisms used by algae to synthesize phenylpropanoids. It would also be important to better understand the distribution and functions of PAL/TAL/PTAL enzymes in both bacteria and fungi, especially in lineages that emerged before land plants, such as the Glomeromycota, with largely unexplored secondary metabolism. The monocot phylogeny includes aquatic and wetland plants such as Spirodela and Acorus spp., but also economically important crops such as vanilla orchids, tulips, agaves, palms, bananas,

Trends in Plant Science, --, Vol. --, No. --

5

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

Trends in Plant Science

Figure 3. Evolutionary History of Aromatic Amino Acid Ammonia-Lyases. The evolution of plants involved significant innovations (;). In particular, the origin of aromatic ammonia-lyases was a key event in plant evolution. Emerging with an antimicrobial role in bacteria, ammonia-lyases were transferred via horizontal gene transfer (HGT) to an early fungal lineage and further acquired by pioneer land plants via arbuscular mycorrhizal symbiosis, probably used initially to synthesize internal signaling molecules [42]. The sequential functions of ammonia-lyases during plant evolution (<) include roles in defense against microorganisms or UV radiation (lignin and flavonoid synthesis), and structural support and water transport (lignin synthesis). PTALs emerged in the monocot grass family (Poaceae) by gene duplication (c. 50–70 Ma) to provide a new or improved function probably related with the complexity of their phenylpropanoid metabolism. Abbreviations: G, Guaiacyl unit of lignin; S, syringyl unit of lignin.

pineapples, asparagus, onions, and major cereals and forage grasses. Among them, some have apparent stems made of leaf stalks (e.g., as bananas) or stems with ’anomalous secondary growth’ (i.e., palms or lianas) that can grow laterally by means of division and enlargement of nonvascular parenchymatic tissue. However, true grasses (Poaceae) are unable to thicken their stems once formed, as they lack the secondary growth typically found in most dicotyledons and gymnosperms. Grass PTALs, along with other grass-specific cell wall-related genes [45,46,47], probably emerged in the Paleocene (50–70 million years ago) during the whole-genome duplication event that separated the Poales from other monocotyledon orders [48]. This event coincides with the warmest and most humid period of the Cenozoic, known as the Pleocene-Eocene Thermal Maximum, which is thought to have stimulated insect population density and accelerated plant species diversification [49,50]. Tyrderived phenylpropanoid biosynthesis might have fulfilled a unique adaptive role in grasses. Grass cell walls show characteristic lignin composition with elevated proportions of S-lignin, cell wall-bound hydroxycinnamates, and the presence of the flavonoid tricin [51,52]. This composition may provide an increased chemical linkage diversity between cell wall polymers that could serve as a barrier to protect the hydro-mineral sap-conducting systems from pathogen attack or offer improved mechanical support properties. Ferulate dimers and oligomers are well-known crosslinking agents between arabinoxylans and lignin in grass cell walls [53], and also exhibit potent antimicrobial activity [54]. Future studies should address the role played by PTAL in the remarkable structural diversity of grass lignins to better understand the exact biological function of this diversity in vivo.

Biological Functions and Regulation PALs (including PTAL) are encoded by a multigene family in most plants and understanding how the different family members are regulated is important for ascribing functions to them. Since being first described in barley in 1961 [55], PALs have become among the most extensively studied enzymes in higher plants because of their key role in the biosynthesis of a large variety of plant-specific phenylpropanoid derivatives. PAL isoforms are expressed differently in various tissues or in response to developmental and environmental factors such as UV irradiation, drought, wounding, or pathogen infection. Such expression patterns have been previously associated with specific metabolic functions

6

Trends in Plant Science, --, Vol. --, No. --

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

(A)

P

(B)

P

Figure 4. Gene Regulatory Networks of PALs and PTALs in Brachypodium distachyon. Coexpression network and proposed biological function (based on gene ontology) of the genes coexpressed with BdPAL (A) and BdPTAL (B). Large database sets were downloaded from PlaNet website (http://aranet.mpimp-golm.mpg.de). The coexpression networks and the Gene Ontology (GO) term enrichment analyses were obtained as described in reference [96]. In the left panels, the genes specifically coexpressed with BdPAL and BdPTAL are highlighted in blue shading in each panel (Tables S1–S4 in the supplemental information online). The transcription factors (TFs) are shown in the networks as diamond boxes. The lignin genes are indicated with black text labels and both BdPAL and BdPTAL are shown shaded in red. The black, (See figure legend continued at the bottom of the next page.)

Trends in Plant Science, --, Vol. --, No. --

7

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

in both dicots [56–59] and monocots [8,60]. Comparative coexpression analyses performed with PAL and PTAL genes from the model grass Brachypodium support the functional differentiation of PALs and PTALs (Figure 4). Genes involved in vesicle transport, and lipid and jasmonate metabolism, were only coexpressed with PALs, whereas the genes coexpressed with PTAL were enriched in biological processes related with abiotic stress, signaling, post-translational modification, and nitrogen, ethylene, and Tyr metabolism. In addition, transcription factors from the LIM and ARR families were specifically coexpressed with PAL, whereas HD-ZIP, ARF, and AP2 families were only coexpressed with PTAL (Tables S1–S4 in the supplemental information online). These observations, along with the suggestion that the distinctive role of PTAL might also be associated with differential cell type-specific expression [9], are consistent with PALs having a more ‘constitutive’ function during normal development for structural lignification in vascular bundles and PTALs being preferentially associated with stress-inducible lignification. In this regard, it should be pointed out that stressinduced lignin is, like the lignin in fibers, often rich in S units [61]. The above findings highlight the importance of obtaining a detailed comparative understanding of the temporal and spatial expression of ammonia-lyases in plants growing under stress conditions. Plant ammonia-lyases exhibit refined regulatory mechanisms at the transcriptional, post-transcriptional, and post-translational levels. PAL gene promoters from different species contain specific target binding sites for several functionally characterized MYB, LIM, ERF, and KNOX transcription factors, which can initiate the tissue-specific and/or stimulus-inducible transcription of PAL genes [62,63]. These AC-rich (or PAL-box) motifs are also found in other phenylpropanoid biosynthetic genes [64], so it is still unclear whether these families of TFs target ammonia-lyases specifically or indirectly through other regulatory genes. Interestingly, the gene that is specifically involved in S-lignin biosynthesis [ferulate/coniferaldehyde 5-hydroxylase (F5H)] is directly regulated by the secondary cell wall master switch NST3 (SND1), which can activate the entire secondary cell wall formation program and regulate the expression of specific MYBs [65]. Considering the fact that modification of PAL or PTAL has different effects on lignin composition [8], it will be interesting to perform deeper promoter studies of PAL and PTAL genes to determine whether different TFs bind differently to PALs and PTALs and also study the interaction of such TFs with other regulators in the network. More research is also needed to understand the post-translational modification of key lignin biosynthetic enzymes, especially in grasses. Early studies indicated that PALs are regulated at the level of both synthesis and turnover [66,67]. In fact, it is currently known that Arabidopsis PALs are regulated by several subunits of the Mediator complex [68,69] and through ubiquitination by Kelch repeat F-box (KFB) proteins via proteolytic degradation [70,71]. Phosphorylation of ammonia-lyases by protein kinases might also be a ubiquitous modification mechanism occurring in higher plants [72,73]. Biochemical and structural studies [30,72] suggest that the phosphorylation site of PAL is located at the end of a flexible helix, connecting the shielding domain to the core domain (Figure 2A,B). This changes the access to the active center of the enzyme. Similar evidence for reversible phosphorylation has recently been reported in poplar COMT [74], another monolignol biosynthetic enzyme with an important role in regulating S-lignin biosynthesis. However, the functional significance of PAL and COMT phosphorylation is still unclear. The flux through the phenylpropanoid pathway is also regulated by chemical intermediates and metabolic channeling. It is well known that ammonia-lyases exhibit product inhibition by intermediates in the same or branch pathways. For example, caffeate inhibits PAL activity in soybean [75], but strongly activates PAL in leaf mustard [76], whereas some flavonoids appear to also serve as metabolic feedback inhibitors of PAL activity in Arabidopsis [77]. Interestingly, the product of the TAL reaction

dark grey, and light grey connectors indicate strong (HRR < 10), medium (HRR < 20), and weak (HRR < 50) strength of the coexpression, respectively. In the right panels, statistical analyses of enriched biological processes obtained from the microarray (MA) terms were performed using Fisher’s exact test (P < 0.05). Abbreviations: CAD, Cinnamyl alcohol dehydrogenase; CCR, cinnamoyl CoA reductase; C3ʹH, 4-coumaroyl shikimate/quinate 3ʹ-hydroxylase; 4CL, 4-hydroxycinnamate:CoA ligase; CHO, carbohydrate; COMT, caffeate/5-hydroxyferulate 3-O-methyltransferase; HCT, 4-hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyltransferase; HRR, highest reciprocal rank; PAL, L-phenylalanine ammonia-lyase; PTAL, bifunctional L-phenylalanine L-/tyrosine ammonia-lyase.

8

Trends in Plant Science, --, Vol. --, No. --

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

(coumarate) inhibits both PAL and TAL activities of PTALs, but has no impact on the activity of monofunctional PALs in Brachypodium [8]. This suggests that PAL and TAL functions in grasses can be differently regulated by specific lignin pathway intermediates. Further research is needed to determine if the levodopa ammonia-lyase activity found in PTALs (Figure 2A) occurs in vivo and if it is inhibited by its own product caffeate or the products from PAL (cinnamate) or TAL (coumarate) reactions. It has also been widely discussed that the enzymes in the phenylpropanoid pathway are regulated through organized multienzyme complexes known as metabolic channels, metabolic compartments, or metabolons [78,79]. Such organization is believed to increase the local concentration of the enzymes and of their substrates to enhance the transfer/turnover efficiency and eliminate the toxicity of intermediate compounds. Most plant CYPs are anchored via their N terminus on the cytoplasmic surface of the endoplasmic reticulum (ER), thus providing appropriate nucleation sites for gathering soluble enzymes and forming metabolons. Other structural components of the ER membrane, including membrane steroid-binding proteins (MSBPs) and reductase/redox proteins [CYP-reductase (ATR2) or cytochrome b5 (CB5D)] may also help to organize and stabilize the membrane-bound metabolon members by serving as scaffold or electron shuttle proteins for the CYPs involved in lignification [80,81] (Figure 5). Ammonia-lyase activity in plants has been proposed to be associated with the ER membrane, and some PAL isoforms colocalize with C4H, the consecutive CYP enzyme in the phenylpropanoid pathway [82]. The observation that some PAL isoforms may be associated differentially with S- and G-lignin biosynthesis [83] led to the suggestion that the production of G and S monolignols may be regulated independently, in a cell type-specific manner [84]. This hypothesis is further supported by the recent observation that downregulation of PTAL in Brachypodium leads to greater reductions in S than G units and also results in a reduction in wall-bound coumarate [8]. Both S-lignin and wall-bound coumarate are preferentially localized in the walls of fiber cells in both dicots and monocots [85,86]. This distribution suggests that fiber and vascular bundle cells in grasses might follow distinct lignification patterns associated with different PAL/TAL cell type-specific expression and/or differential availabilities of Phe and Tyr (Figure 5). In this hypothetical working model, a cytosolic route to lignin, likely to be involved in abiotic stress responses (Figure 4), is first initiated by PTAL and C3H/APX [8,9] and links S-lignin deposition in fiber cells with stress-induced reactive oxygen species, specifically hydrogen peroxide. This model is partially supported by recent mutant studies in rice (Oryza sativa) showing that ER-associated p-coumaroyl quinate/shikimate 3ʹ-hydroxylase (C3ʹH) and F5H are primarily involved in the conventional biosynthesis of nonacylated G/S-lignins but not in the biosynthesis of grass-specific p-coumaroylated S-lignins [87,88]. The precise physiological functions of PTAL and the regulation of this metabolic response in a cell-specific manner remain to be explored.

Biotechnological Applications Plant ammonia-lyases have applications both as isolated recombinant enzymes and in bioengineering/synthetic biology. Although PTALs are present in some higher plants (Figure S1 in the supplemental information online), the commercial sources of enzyme have only been the PTAL of the fungus Rhodotorula and bacteria of the genus Rhodobacter [89]. The reverse reaction catalyzed by PAL can be achieved under certain physiological conditions such as high ammonia concentrations. Actually, this reaction has been widely used to convert cinnamate into Phe, as a precursor for industrial production of the artificial sweetener aspartame [41]. However, similar reverse reactions using PTALs for the production of Tyr or DOPA from coumarate or caffeate have not been explored to date. Tyr is itself a valuable compound used as a common dietary supplement. It is also a precursor for high-value compounds such as DOPA and carbidopa, used for the treatment of Parkinson’s disease, or melanins, used as UV blockers, cation exchangers, semiconductors, and drug carriers [90]. Both the forward and reverse PTAL reactions are of interest for multiple industrial applications. Bacterial and fungal PTALs have been previously used for the production of aromatic compounds such as coumarate, stilbenes, flavonoids, curcuminoids, cinnamoyl anthranilates, and plastic, cosmetic, and adhesive precursors [89,91–93]. Screening for efficient TAL enzymes among monocot PTALs within the plant kingdom would be valuable, as in most cases the TAL reaction is the limiting step in

Trends in Plant Science, --, Vol. --, No. --

9

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

(A)

(B) (C)

Trends in Plant Science

Figure 5. Hypothetical Model of Cell Type-Specific Lignification in Grasses. The model depicts suggested differences between two pathways for lignin formation from the aromatic amino acids L-tyrosine and L-phenylalanine associated with sclerenchyma fibers (A) and tracheary elements (B), respectively. The figure shows a partly hypothetical, fully cytosolic route (A) involving phenolic acids preferentially associated with the biosynthesis of S-units of lignin in fiber cells; this would require the presence of a yet-to-be discovered non-membrane-bound ferulate 5-hydroxylase (F5H) enzyme. This cytosolic pathway, initiated by bifunctional phenylalanine/tyrosine ammonia-lyase (PTAL) and coumarate 3-hydroxylase/ascorbate peroxidase (C3H/APX) [8,9], links phenylpropanoid metabolism with stress-induced reactive oxygen species (ROS) through hydrogen peroxide (H202) detoxification and ascorbate–glutathione metabolism and is likely to be involved in abiotic stress responses. This model forms a working hypothesis for further studies. Panel (B) shows the currently accepted pathway to H, G, and S monolignols in dicots. Panel (C) shows a simplified diagram of the esters and acids routes to monolignol biosynthesis. Lignin enzyme (cofactors): PAL, PTAL, and CSE (no cofactor required); C3H (ascorbate); C4H, C3ʹH, F5H, CAD, and CCR (NADPH or NADH); COMT and CCoAOMT (S-adenosyl L-methionine, SAM). Abbreviations: ATR2, NADPH-dependent cytochrome P450 oxidoreductase; CAD, cinnamyl alcohol dehydrogenase; CB5D, NADH-cytochrome b5 reductase isoform D serving as electron donors to CYPs involved in lignification [80,81]; CCoAOMT, caffeoyl-CoA 3-O-methyltransferase; CCR, cinnamoyl-CoA reductase; C3ʹH, 4-coumaroyl shikimate/quinate 3-hydroxylase; C3H/APX, bifunctional 4-coumarate 3-hydroxylase/cytosolic ascorbate peroxidase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; COMT, caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase; CSE, caffeoyl shikimate esterase; CYP, cytochrome P450; ER, endoplasmic reticulum; F5H, ferulate/coniferaldehyde 5-hydroxylase; G, guaiacyl unit of lignin; H, p-hydroxyphenyl unit of lignin; HCT, hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase; MSBP, membrane steroid-binding protein serving as a scaffold to cluster monolignol CYPs; NH3+, N terminus of P450; PAL, L-phenylalanine ammonia-lyase; PTAL, bifunctional L-phenylalanine/L-tyrosine ammonia-lyase; S, syringyl unit of lignin.

synthetic biology approaches to Tyr-derived phenylpropanoids [93,94]. To enhance the capacity for Tyr synthesis as a precursor to produce Tyr-derived aromatic products, some of the rate-limiting enzymes in Tyr biosynthesis such as arogenate dehydrogenase (ADH) or Tyr aminotransferase (TyrAT) could also be considered for overexpression. Overexpressing PTAL in dicot plants not naturally harboring this enzyme is expected to increase lignin deposition and change monolignol composition, whereas downregulation of endogenous PTALs in grasses is expected to reduce lignin levels

10

Trends in Plant Science, --, Vol. --, No. --

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

and increase the bioavailability of Tyr. The latter may be of interest for introducing/engineering the biosynthesis of Tyr-derived compounds such as betalains, plastoquinones, catecholamines, or tocopherols in major food crops.

Concluding Remarks Plant PALs have a key role in mediating and coordinating carbon flux from primary to secondary metabolism, which is of primary importance for plants to cope with varying environmental conditions for adaptation and optimal growth. Recent studies indicate that true grasses (Poaceae) likewise use PTALs with similar but distinct biological functions. Understanding the precise mechanisms of regulation and functions of both plant aromatic ammonia-lyases will facilitate strategies to regulate phenylpropanoid deposition in major food crops, which could significantly enhance plant productivity for the transition toward a bioeconomy era. A first step in this direction would be to better understand the protein structure, substrate selectivity, and mechanisms of ammonia elimination. An important step towards elucidation of the precise biological functions of PALs and PTALs can be achieved by studying the unexplored distribution and roles of ammonia-lyases in algal, bacterial, and fungal lineages that emerged before the land plants and by examining the differential cell-specific gene regulatory networks at the transcriptional and post-transcriptional levels between monocot and dicot plants. Finally, identifying the specific multienzyme complexes containing PALs and PTALs, which have been widely discussed in the literature, often without direct experimental evidence, could provide a basis to engineer novel metabolic pathways in plants or microbes with innovative biotechnological applications (see Outstanding Questions).

Outstanding Questions What is the exact mechanism of ammonia elimination and what are the key amino acid residues that control TAL activity of PTALs? What are the biological functions of PTALs and what are their roles in the structural diversity of grass lignins? Are PALs and PTALs regulated by different sets of transcription factors? If so, what is their interaction with other regulators in the lignin/ phenylpropanoid network? What is the cellular and tissue localization of PAL and PTAL? What are the participating enzymes in the lignin metabolons and their extension? Are plant PTALs suitable for successfully engineering new metabolic pathways in other organisms for new biotechnological applications?

Acknowledgments This manuscript is dedicated to the memory of Prof. Ignacio Zarra. We are grateful to Dr Xiaolan Rao for help with the coexpression network, Dr Xiaoqiang Wang for advice on protein molecular modelling, and Dr Maite Docampo-Palacios for discussion of chemical reaction schemes. This work was supported by a grant from The University of Santiago de Compostela (to J.B.), and the University of North Texas and the Center for Bioenergy Innovation (Oak Ridge National Laboratory), a US Department of Energy (DOE) Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. This manuscript has been coauthored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the US Department of Energy.

Supplemental Information Supplemental information associated with this article can be found online at https://doi.org/10.1016/ j.tplants.2019.09.011. References 1. Wu, Z. et al. (2014) Molecular evolution and functional characterisation of an ancient phenylalanine ammonia-lyase gene (NnPAL1) from Nelumbo nucifera: novel insight into the evolution of the PAL family in angiosperms. BMC Evol. Biol. 14, 100 2. Hart, P.H. et al. (2019) Exposure to ultraviolet radiation in the modulation of human diseases. Annu. Rev. Pathol. 14, 55–81 3. Nielsen, L.N. et al. (2018) Glyphosate has limited short-term effects on commensal bacterial community composition in the gut environment due to sufficient aromatic amino acid levels. Environ. Pollut. 233, 364–376 4. Maeda, H. and Dudareva, N. (2012) The shikimate pathway and aromatic amino acid biosynthesis in plants. Annu. Rev. Plant Biol. 63, 73–105 5. Yoo, H. et al. (2013) An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine: phenylpyruvate aminotransferase. Nat. Commun. 4, 2833

6. Vanholme, R. et al. (2013) Caffeoyl shikimate esterase (CSE) is an enzyme in the lignin biosynthetic pathway in Arabidopsis. Science 341, 1103–1106 7. Nair, R.B. et al. (2004) The Arabidopsis thaliana reduced epidermal fluorescence1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis. Plant Cell 16, 544–554 8. Barros, J. et al. (2016) Role of bifunctional ammonialyase in grass cell wall biosynthesis. Nat. Plants 2, 16050 9. Barros, J. et al. (2019) 4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase. Nat. Commun. 10, 1994 10. Sun, P. et al. (2019) My way: noncanonical biosynthesis pathways for plant volatiles. Trends Plant Sci. 21, 884–894 11. Renault, H. et al. (2017) A phenol-enriched cuticle is ancestral to lignin evolution in land plants. Nat. Commun. 8, 14713 12. Deng, Y. and Shanfa, L. (2017) Biosynthesis and regulation of phenylpropanoids in plants. Crit. Rev. Plant Sci. 36, 257–290

Trends in Plant Science, --, Vol. --, No. --

11

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

13. Dreßen, A. et al. (2017) Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2): a potent MIOenzyme for the synthesis of non-canonical aromatic alpha-amino acids. Part I: comparative characterization to the enzymes from Petroselinum crispum (PcPAL1) and Rhodosporidium toruloides (RtPAL). J. Biotechnol. 258, 148–157 14. Kyndt, J.A. et al. (2002) Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein. FEBS Lett. 512, 240–244 15. Berner, M. et al. (2006) Genes and enzymes involved in caffeic acid biosynthesis in the actinomycete Saccharothrix espanaensis. J. Bacteriol. 188, 2666– 2673 16. Watts, K.T. et al. (2006) Discovery of a substrate selectivity switch in tyrosine ammonia-lyase, a member of the aromatic amino acid lyase family. Chem. Biol. 13, 1317–1326 17. Goldson-Barnaby, A. and Scaman, C.H. (2013) Purification and characterization of phenylalanine ammonia lyase from Trichosporon cutaneum. Enzyme Res. 2013, 670702 18. Jun, S.-Y. et al. (2018) Biochemical and structural analysis of substrate specificity of a phenylalanine ammonia-lyase. Plant Physiol. 176, 1452–1468 19. Louie, G.V. et al. (2006) Structural determinants and modulation of substrate specificity in phenylalanine-tyrosine ammonia-lyases. Chem. Biol. 13, 1327–1338 20. Hsieh, L.S. et al. (2010) Cloning, expression, sitedirected mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii. Phytochem. 71, 1999–2009 21. Pilba´k, S. et al. (2006) The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases. FEBS J. 273, 1004– 1019 22. Reichert, A.I. et al. (2009) Phenylalanine ammonialyase (PAL) from tobacco (Nicotiana tabacum): characterization of the four tobacco PAL genes and active heterotetrameric enzymes. Biochem. J. 424, 233–242 23. Hyun, M.W. et al. (2011) Fungal and plant phenylalanine ammonia-lyase. Mycobiology 39, 257–265 24. Ro¨ther, D. et al. (2002) An active site homology model of phenylalanine ammonia-lyase from P. crispum. Eur. J. Biochem. 269, 3065–3075 25. Pilba´k, S. et al. (2012) Mechanism of the tyrosine ammonia lyase reaction—tandem nucleophilic and electrophilic enhancement by a proton transfer. Chem. Eur. J. 18, 7793–7802 26. Lovelock, S.L. et al. (2014) Phenylalanine ammonia lyase catalyzed synthesis of amino acids by an MIO-cofactor independent pathway. Angew. Chem. 126, 4740–4744 27. Pinto, G.P. et al. (2015) New insights in the catalytic mechanism of tyrosine ammonia-lyase given by QM/MM and QM cluster models. Arch. Biochem. Biophys. 582, 107–115 28. Poppe, L. and Re´tey, J. (2005) Friedel–Crafts-type mechanism for the enzymatic elimination of ammonia from histidine and phenylalanine. Ange. Chem. Int. Ed. 44, 3668–3688 29. Weiser, D. et al. (2015) Phenylalanine ammonia-lyase-catalyzed deamination of an acyclic amino acid: enzyme mechanistic studies aided by a novel microreactor filled with magnetic nanoparticles. ChemBioChem 16, 2283–2288 30. Ritter, H. and Schulz, G.E. (2004) Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase. Plant Cell 16, 3426–3436

12

Trends in Plant Science, --, Vol. --, No. --

31. Calabrese, J.C. et al. (2004) Crystal structure of phenylalanine ammonia lyase: multiple helix dipoles implicated in catalysis. Biochemistry 43, 11403–11416 32. Mu¨ller, I. (2017) Guidelines for the successful generation of protein–ligand complex crystals. Acta Cryst. D 73, 79–92 33. Harholt, J. et al. (2016) Why plants were terrestrial from the beginning. Trends Plant Sci. 21, 96–101 34. Renault, H. (2019) Harnessing lignin evolution for biotechnological applications. Curr. Opin. Biotechnol. 56, 105–111 35. Stafford, H.A. (1991) Flavonoid evolution: an enzymic approach. Plant Phys 96, 680–685 36. Ligrone, R. et al. (2008) Immunocytochemical detection of lignin-related epitopes in cell walls in bryophytes and the charalean alga Nitella. Plant Syst. Evol. 270, 257–272 37. Moffitt, M.C. et al. (2007) Discovery of two cyanobacterial phenylalanine ammonia lyases: kinetic and structural characterization. Biochemistry 46, 1004–1012 38. Xiang, L. and Moore, B.S. (2005) Biochemical characterization of a prokaryotic phenylalanine ammonia lyase. J. Bacteriol. 187, 4286–4289 39. Williams, J.S. et al. (2005) The gene stlA encodes a phenylalanine ammonia-lyase that is involved in the production of a stilbene antibiotic in Photorhabdus luminescens TT01. Microbiology 151, 2543–2550 40. Bandoni, R.J. et al. (1968) Phenylalanine and tyrosine ammonia-lyase activity in some Basidiomycetes. Phytochemistry 7, 205–207 41. MacDonald, M.C. et al. (2016) Rhodotorula glutinis phenylalanine/tyrosine ammonia lyase enzyme catalyzed synthesis of the methyl ester of parahydroxycinnamic acid and its potential antibacterial activity. Front. Microbiol. 7, 281 42. Emiliani, G. et al. (2009) A horizontal gene transfer at the origin of phenylpropanoid metabolism: a key adaptation of plants to land. Biol. Direct 4, 1 43. Martone, P.T. et al. (2009) Discovery of lignin in seaweed reveals convergent evolution of cell-wall architecture. Curr. Biol. 19, 169–175 44. Sørensen, I. et al. (2011) The charophycean green algae provide insights into the early origins of plant cell walls. Plant J. 68, 201–211 45. Petrik, D.L. et al. (2014) p-Coumaroyl-CoA: monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon. Plant J. 77, 713–726 46. Bhatia, R. et al. (2017) Genetic engineering of grass cell wall polysaccharides for biorefining. Plant Biotechnol. J. 15, 1071–1092 47. Lam, P.Y. et al. (2019) Recruitment of specific flavonoid B-ring hydroxylases for two independent biosynthesis pathways of flavone-derived metabolites in grasses. New Phytol. 223, 204–219 48. Jiao, Y. et al. (2014) Integrated syntenic and phylogenomic analyses reveal an ancient genome duplication in monocots. Plant Cell 26, 2792–2802 49. Currano, E.D. et al. (2008) Sharply increased insect herbivory during the Paleocene–Eocene Thermal Maximum. Proc. Natl. Acad. Sci. U. S. A. 105, 1960– 1964 50. Cai, L. et al. (2019) Widespread ancient whole-genome duplications in Malpighiales coincide with Eocene global climatic upheaval. New Phytol. 221, 565–576 51. Hassan, A.S. and Burton, R.A. (2018) Role, importance and biosynthesis of cell wall-bound phenolic acids in cereals. Annu. Plant Rev. 3, 1–30 52. Lan, W. et al. (2016) Tricin-lignins: occurrence and quantitation of tricin in relation to phylogeny. Plant J. 88, 1046–1057 53. Waterstraat, M. and Bunzel, M. (2018) A multi-step chromatographic approach to purify radically

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

54. 55.

56.

57.

58.

59.

60.

61.

62.

63.

64. 65.

66.

67. 68. 69.

70.

71.

generated ferulate oligomers reveals naturally occurring 5-5/8-8 (cyclic)-, 8-8 (noncyclic)/8-O-4-, and 5-5/8-8 (noncyclic)-coupled dehydrotriferulic acids. Front. Chem. 6, 190 Piotrowski, J.S. et al. (2015) Plant-derived antifungal agent poacic acid targets b-1, 3-glucan. Proc. Natl. Acad. Sci. U. S. A. 112, E1490–E1497 Koukol, J. and Conn, E.E. (1961) The metabolism of aromatic compounds in higher plants IV. Purification and properties of the phenylalanine deaminase of Hordeum vulgare. J. Biol. Chem. 236, 2692–2698 Olsen, K.M. et al. (2008) Differential expression of four Arabidopsis PAL genes; PAL1 and PAL2 have functional specialization in abiotic environmentaltriggered flavonoid synthesis. J. Plant Physiol. 165, 1491–1499 Rohde, A. et al. (2004) Molecular phenotyping of the pal1 and pal2 mutants of Arabidopsis thaliana reveals far-reaching consequences on phenylpropanoid, amino acid, and carbohydrate metabolism. Plant Cell 16, 2749–2771 Huang, J. et al. (2010) Functional analysis of the Arabidopsis PAL gene family in plant growth, development, and response to environmental stress. Plant Physiol. 153, 1526–1538 Shine, M.B. et al. (2016) Cooperative functioning between phenylalanine ammonia lyase and isochorismate synthase activities contributes to salicylic acid biosynthesis in soybean. New Phytol. 212, 627–636 Cass, C.L. et al. (2015) Effects of phenylalanine ammonia lyase (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium. J. Exp. Bot. 66, 4317–4335 Rao, X. et al. (2017) Dynamic changes in transcriptome and cell wall composition underlying brassinosteroid-mediated lignification of switchgrass suspension cells. Biotechnol. Biofuels 10, 266 Zhang, X. and Liu, C.J. (2015) Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoids. Mol. Plant 8, 17–27 Guo, W. et al. (2016) An ethylene response-related factor, GbERF1-like, from Gossypium barbadense improves resistance to Verticillium dahliae via activating lignin synthesis. Plant Mol. Biol. 91, 305–318 Kawaoka, A. et al. (2000) Functional analysis of tobacco LIM protein Ntlim1 involved in lignin biosynthesis. Plant J. 22, 289–301 Zhao, Q. et al. (2010) Syringyl lignin biosynthesis is directly regulated by a secondary cell wall master switch. Proc. Natl. Acad. Sci. U. S. A. 107, 14496– 14501 Edwards, K. et al. (1985) Rapid transient induction of phenylalanine ammonia-lyase mRNA in elicitortreated bean cells. Proc. Natl. Acad. Sci. U. S. A. 82, 6731–6735 Bolwell, G.P. et al. (1985) L-Phenylalanine ammonia-lyase from Phaseolus vulgaris. Eur. J. Biochem. 149, 411–419 Bonawitz, N.D. et al. (2014) Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant. Nature 509, 376–380 Dolan, W.L. et al. (2017) Mediator complex subunits MED2, MED5, MED16, and MED23 genetically interact in the regulation of phenylpropanoid biosynthesis. Plant Cell 29, 326903285 Zhang, X. et al. (2013) Arabidopsis Kelch repeat F-box proteins regulate phenylpropanoid biosynthesis via controlling the turnover of phenylalanine ammonialyase. Plant Cell 25, 4994–5010 Yu, S.I. et al. (2019) Post-translational and transcriptional regulation of phenylpropanoid

72.

73.

74.

75.

76. 77.

78. 79. 80. 81. 82.

83.

84. 85.

86. 87.

88.

89.

90.

biosynthesis pathway by Kelch repeat F-box protein SAGL1. Plant Mol. Biol. 99, 135–148 Bolwell, G.P. (1992) A role for phosphorylation in the down-regulation of phenylalanine ammonia-lyase in suspension-cultured cells of French bean. Phytochemistry 31, 4081–4086 Cheng, S.H. et al. (2001) Molecular identification of phenylalanine ammonia-lyase as a substrate of a specific constitutively active Arabidopsis CDPK expressed in maize protoplasts. FEBS Lett. 503, 185–188 Wang, J.P. et al. (2015) Phosphorylation is an on/off switch for 5-hydroxyconiferaldehyde Omethyltransferase activity in poplar monolignol biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 112, 8481– 8486 Bubna, G.A. et al. (2011) Exogenous caffeic acid inhibits the growth and enhances the lignification of the roots of soybean (Glycine max). J. Plant Physiol. 168, 1627–1633 Lim, H.W. et al. (1997) Purification and properties of phenylalanine ammonia-lyase from leaf mustard. Mol. Cells. 7, 715–720 Yin, R. et al. (2012) Feedback inhibition of the general phenylpropanoid and flavonol biosynthetic pathways upon a compromised flavonol-3-O-glycosylation. J. Exp. Bot. 63, 2465–2478 Winkel, B.S. (2004) Metabolic channeling in plants. Annu. Rev. Plant Biol. 55, 85–107 Knudsen, C. et al. (2018) Dynamic metabolic solutions to the sessile life style of plants. Nat. Prod. Rep. 35, 1140–1155 Gou, M. et al. (2018) The scaffold proteins of lignin biosynthetic cytochrome P450 enzymes. Nat. Plants 4, 299–310 Gou, M. et al. (2019) Cytochrome b5 is an obligate electron shuttle protein for syringyl lignin biosynthesis in Arabidopsis. Plant Cell 31, tpc.00778. Achnine, L. et al. (2004) Colocalization of L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis. Plant Cell 16, 3098– 3109 Sewalt, V.J. et al. (1997) Reduced lignin content and altered lignin composition in transgenic tobacco down-regulated in expression of L-phenylalanine ammonia-lyase or cinnamate 4-hydroxylase. Plant Physiol. 115, 41–50 Dixon, R.A. et al. (2002) The phenylpropanoid pathway and plant defence—a genomics perspective. Molec. Plant Pathol. 3, 371–390 Nakashima, J. et al. (2008) Multi-site genetic modification of monolignol biosynthesis in alfalfa (Medicago sativa): effects on lignin composition in specific cell types. New Phytol. 179, 738–750 Ralph, J. et al. (1994) Identification and synthesis of new ferulic acid dehydrodimers present in grass cell walls. J. Chem. Soc. 1, 3485–3498 Takeda, Y. et al. (2018) Downregulation of p-COUMAROYL ESTER 3-HYDROXYLASE in rice leads to altered cell wall structures and improves biomass saccharification. Plant J. 95, 796–811 Takeda, Y. et al. (2019) Lignin characterization of rice CONIFERALDEHYDE 5-HYDROXYLASE loss-of-function mutants generated with the CRISPR/ Cas9 system. Plant J. 97, 543–554 Kong, J.Q. (2015) Phenylalanine ammonia-lyase, a key component used for phenylpropanoids production by metabolic engineering. RSC Adv. 5, 62587–62603 Lu¨tke-Eversloh, T. et al. (2007) Perspectives of biotechnological production of L-tyrosine and its applications. Appl. Microbiol. Biotechnol. 77, 751–762

Trends in Plant Science, --, Vol. --, No. --

13

Please cite this article in press as: Barros and Dixon, Plant Phenylalanine/Tyrosine Ammonia-lyases, Trends in Plant Science (2019), https:// doi.org/10.1016/j.tplants.2019.09.011

Trends in Plant Science

91. Jendresen, C.B. et al. (2015) Novel highly active and specific tyrosine ammonia-lyases from diverse origins enable enhanced production of aromatic compounds in bacteria and yeast. Appl. Environ. Microbiol. 81, 4458–4476 92. Katsuyama, Y. et al. (2008) Production of curcuminoids by Escherichia coli carrying an artificial biosynthesis pathway. Microbiology 154, 2620–2628 93. Eudes, A. et al. (2013) Production of hydroxycinnamoyl anthranilates from glucose in Escherichia coli. Microb. Cell Fact 12, 1

14

Trends in Plant Science, --, Vol. --, No. --

94. Lin, Y. and Yan, Y. (2012) Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex. Microb. Cell Fact. 11, 1 95. Parmeggiani, F. et al. (2018) Synthetic and therapeutic applications of ammonialyases and aminomutases. Chem. Rev. 118, 73–118 96. Mutwil, M. et al. (2011) PlaNet: combined sequence and expression comparisons across plant networks derived from seven species. Plant Cell 23, 895–910