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Keywords:

  • enzymology;
  • evolution;
  • protein function;
  • protein structure

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References

Mammalian phenylalanine hydroxylase (PAH) catalyzes the rate-limiting step in the phenylalanine catabolism, consuming about 75% of the phenylalanine input from the diet and protein catabolism under physiological conditions. In humans, mutations in the PAH gene lead to phenylketonuria (PKU), and most mutations are mainly associated with PAH misfolding and instability. The established treatment for PKU is a phenylalanine-restricted diet and, recently, supplementation with preparations of the natural tetrahydrobiopterin cofactor also shows effectiveness for some patients. Since 1997 there has been a significant increase in the understanding of the structure, catalytic mechanism, and regulation of PAH by its substrate and cofactor, in addition to improved correlations between genotype and phenotype in PKU. Importantly, there has also been an increased number of studies on the structure and function of PAH from bacteria and lower eukaryote organisms, revealing an additional anabolic role of the enzyme in the synthesis of melanin-like pigments. In this review, we discuss these recent studies, which contribute to define the evolutionary adaptation of the PAH structure and function leading to sophisticated regulation for effective catabolic processing of phenylalanine in mammalian organisms. © 2013 IUBMB Life 65(4):341–349, 2013.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References

Phenylalanine hydroxylase (PAH, EC 1.14.16.1) catalyzes the conversion of L-phenylalanine (L-Phe) to L-tyrosine (L-Tyr) by para-hydroxylation of the aromatic side-chain. In mammals, this tetrahydrobiopterin (BH4)-dependent reaction is the initial and rate-limiting step in the degradation of excess L-Phe from dietary proteins, where L-Tyr is further degraded to products that feed into the citric acid cycle (Fig. 1). L-Tyr is thus a non-essential amino acid in PAH-containing organisms, where it is used for protein synthesis or as precursor for neurotransmitters and other L-Tyr derivatives. The function of PAH in mammals has been studied since the end of the 1950s (see ref.1 for a review of the earlier studies). PAH is primarily present in the liver, where removal of excess L-Phe prevents the neurotoxic effect of hyperphenylalaninemia (HPA). However, L-Phe is also an essential proteinogenic amino acid and it is important to avoid that it is fully catabolized. To accomplish this dual role of preserving, yet removing excess L-Phe effectively, mammalian PAH has developed several regulatory mechanisms and a specific structure—the framework through which the regulatory properties are exerted. In recent years, there have been important advances in the elucidation of the catalytic mechanism and structure–function relationships (2–4), but less is known about the structure–regulation relationships (5). PAH is also present in non-mammalian eukaryote organisms and some bacteria, and notably in the last decade several studies have investigated PAH from these sources. In addition to catabolic degradation (6, 7), PAH from bacteria and non-mammalian eukaryote organisms has a prominent anabolic role contributing to the synthesis of melanin-type pigments (8–11) (Fig. 1). This review focuses on the present understanding of the structure, function, and regulation of mammalian PAH, and includes a comparative discussion of the enzyme from different organisms along an evolutionary perspective.

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Figure 1. The PAH reaction and complete L-Phe catabolism (black pathway). In blue (and squared gray background), the abnormal metabolism of L-Phe in phenylketonuria, through transamination and decarboxylation of accumulated L-Phe. In red (and round grey background), the production of pyomelanin from homogentisate occurring in some bacteria. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Phenylketonuria

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References

Dysfunctional PAH leads to increased concentration of L-Phe in the blood and the appearance in urine of metabolites that arise from the transamination of L-Phe to phenylpyruvate (Fig. 1). This is the hallmark of the HPAs, of which classic phenylketonuria (PKU) (OMIM 261600) is the most severe form with plasma L-Phe levels >1,200 μM (12). The accumulation of L-Phe and the subsequent disturbance in brain neurotransmitters lead to neurological symptoms including mental retardation, purposeless movements, and depression (for a complete list of symptoms, see www.omim.org). The dietary intake of L-Phe must therefore be strictly controlled in PKU patients (12–14).

PKU is inherited as an autosomal recessive disorder, with more than 500 disease-causing mutations (www.pahdb.mcgill.ca and www.biopku.org). In vitro studies of mutant proteins have revealed the kinetic and conformational defects caused by the mutations. PKU appears largely as a misfolding disease where loss of enzymatic function is caused mainly by folding defects leading to decreased stability, and PKU is considered a paradigm for misfolding loss-of-function genetic disorders (13, 15, 16). The molecular basis for the neurological symptoms is not completely understood, but saturation by L-Phe of the LAT-1 transporter at the blood–brain barrier and defective myelination seem critical. Recently, a new amyloidosis-like etiology for PKU has been suggested, since L-Phe has been shown to self-assemble into toxic fibrils (17).

While displaying highest affinity for L-Phe, LAT-1 is also the only route for eight other large neutral amino acids (LNAAs) into the brain (18). LNAAs are important for cerebral protein synthesis, and L-Tyr and L-Tryptophan (L-Trp) are also precursors for neurotransmitters. Saturation of LAT-1 by elevated levels of L-Phe thus creates an imbalance in both neurotransmitter- and protein synthesis, including hypomyelination. Consequently, supplementation with LNAAs is one treatment strategy currently investigated, in addition to enzymatic therapy with pegylated phenylalanine ammonia lyase, gene therapy, pharmacological chaperones and the already approved supplementation with the cofactor BH4 (for a review see14). A number of studies have contributed to reveal the most recurrent genotypes associated with BH4-responsive PKU and the molecular mechanisms behind the corrective effects of BH4 (12, 19).

Aromatic Amino Acid Hydroxylases

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References

PAH is a member of the aromatic amino acid hydroxylase (AAAH) enzyme family. The AAAHs share a requirement for a catalytic non-heme ferrous iron, BH4 as cofactor, and molecular oxygen as additional substrate. The mammalian genes encode PAH, tyrosine hydroxylase (TH), and two tryptophan hydroxylases (TPH1 and TPH2), which are named after their specific amino acid substrates. The products of TH (L-3,4-dihydroxyphenylalanine (L-DOPA)) and the TPHs (5-hydroxytryptophan) are precursors for important neurotransmitters and hormones in the brain and the neuroendocrine system (2). The AAAHs show high sequence identity (Fig. 2A), similar structure, and presumed similar catalytic mechanism (2, 3). Metazoans have three or four AAAHs-encoding genes, but only a single gene has so far been found in protozoans, such as Leishmania major (20), and in the slime mold Dictyostelium discoideum (21). These single AAAHs have been identified as PAHs (20, 21). Moreover, PAH is the only AAAH found in bacteria. In fact, based on the homology between the regulatory AAAH domains and prephenate dehydratase, Gjetting et al. proposed that bacterial PAH and prephenate dehydratase were the precursors of multi-domain AAAHs (22). A role of PAH as the ancestral function in the AAAH family appears reasonable since the TH and TPH functions are likely to be of increasing importance in pluricellular organisms. As will be shown in the next sections, PAH has evolved its structural organization to enable sophisticated regulation, thereby adapting to the emerging metabolic and neurological needs of high-complexity organisms.

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Figure 2. Domain organization and structure of PAH. A: Alignment of human AAAHs and PAHs from different organisms. Bars represent gaps (white), non-identical residues (gray), residues identical in ≥60% of the sequences (dark gray), and residues identical in all the sequences (black). RD, regulatory domain; CD, catalytic domain; OD, oligomerization domain. The highest homology (80%) is encountered in the catalytic domains. B: Composite model of full-length tetrameric human PAH prepared with PDBs 2PHM and 2PAH. Inset, domain organization of each subunit. C: The structure of the ternary PAH · Fe(II) · BH4 · L-Phe complex, based on PDB 1MMK, with L-Phe modeled at the 3-(2-thienyl)-L-alanine binding site. D: The crystal structure of PAH from C. violaceum PAH (PDB 1LTV). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The Phenylalanine Hydroxylase System

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References

As for the other AAAHs, PAH catalyzes the hydroxylation of its substrate by incorporation of one oxygen atom into the aromatic ring, and the final reaction also includes the reduction of the second oxygen atom to water using two electrons supplied by BH4. The cofactor BH4 functions as a co-substrate that is also hydroxylated at each turnover to pterin-4a-carbinolamine (4a-OH-BH4), with consequent dissociation from the enzyme (23) (Fig. 1). The dehydration and reduction of 4a-OH-BH4 back to BH4 is catalyzed sequentially by pterin carbinolamine dehydratase, which dehydrates 4a-OH-BH4 to dihydrobiopterin quinonoid (q-BH2), and the NADH-dependent dihydropteridine reductase, respectively (23). These two enzymes are therefore considered as part of the PAH system, making the degradation of L-Phe sensitive to defects in several genes. If q-BH2 rearranges to BH2, reduction to BH4 can be catalyzed by dihydrofolate reductase (23).

Structure of Mammalian PAH

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References

Mammalian PAH is a homo-tetrameric enzyme of 50 kDa subunits. The structure of a full-length mammalian PAH has not yet been solved, but truncated PAH structures are available (Table 1). A composite model of full-length tetrameric PAH can be prepared based on crystal structures from dimeric truncated rat PAH (PDB 2PHM) and tetrameric human PAH (hPAH) (PDB 2PAH) (Fig. 2B). Although only the BH4-responsive PKU mutant A313T-PAH has been amenable to crystal structure determination (19), the available structures have been crucial for correlating genotypes and phenotype in PKU, defining hotspots for enzyme destabilization (4, 15).

Table 1. Representative PAH structures solved by crystallography
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Each PAH subunit is composed of an N-terminal regulatory domain (residues 1–110), a central catalytic domain (residues 111–410), and a C-terminal oligomerization domain (residues 411–452) (24, 25) (Fig. 2B). The N-terminal regulatory domain is classified as an ACT-domain, a regulatory module present in several proteins, which often dimerizes and binds amino acids (26). This domain is flexibly attached to the catalytic domain via a hinge region (Arg111–Thr117), but establishes extensive contacts with the catalytic domain of the adjacent subunit within the dimer. The regulatory domain is necessary for manifestation of the regulatory properties, such as activation by L-Phe, and it is still debated whether or not it includes an allosteric binding-site for L-Phe. In support of the existence of a regulatory site, Gjetting et al. remarked that the regulatory domain presents two motifs, GAL (residues 46–48 in hPAH) and (I/L)ESRP (residues 65–69), which are involved in L-Phe binding in a bacterial prephenate dehydratase, and demonstrated that isolated regulatory domains of hPAH actually form a dimer and bind L-Phe (22). However, whether this intersubunit regulatory binding site is functional in full-length PAH, where (according to the available structural models) regulatory domains are physically separated (Fig. 2B), has not been convincingly demonstrated (see also next section). Preceding the ACT-domain is an intrinsic autoregulatory sequence (residues 1–33 in hPAH) that extends over the active site (24) and limits L-Phe access, especially when its physiological cofactor (6R)-BH4 is bound (24, 27–29). The highly mobile N-terminal (residues 1–18) of the intrinsic autoregulatory sequence is not seen in the crystal structure, and conformational information has been obtained by molecular modeling (30).

The catalytic domain contains the binding sites for iron, cofactor, and substrate. At the active site iron binds to two histidines (His285 and His290 in hPAH) and a glutamate (Glu330 in hPAH) (Fig. 2C). Structures with bound L-Phe analogues 3-(2-thienyl)-L-alanine or L-norleucine, and reduced or oxidized cofactor at the active site (Table 1; Fig. 2C) have provided the molecular frames to elucidate the AAAH mechanism (2, 3, 31). Formation of the high-spin Fe(IV) hydroxylating species—identified experimentally in the catalytic cycles of both TH (32) and PAH from Chromobacterium violaceum (33)—is a crucial step in the reaction.

The oligomerization domain starts by an antiparallel-sheet (residues 411–414, 421–424), responsible for dimerization, followed by a 40 Å long-helix (428–452) that mediates tetramerization through domain swapping and antiparallel coiled-coil formation with the other monomers (25) (Fig. 2B).

Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References

Several mechanisms act together to tightly control the activity of mammalian PAH, and some short-term effects known to play a physiological role are caused by L-Phe, the natural cofactor (6R)-BH4, and phosphorylation at Ser16 by cAMP-dependent protein kinase. Liver PAH from different mammals displays the same physical and regulatory properties as the human enzyme, although there are some differences in the activation constants and notably the extent of the activation by L-Phe (5).

The activity of tetrameric mammalian PAH—when assayed with 6R-BH4—is activated by incubation with L-Phe and responds with positive cooperativity toward increasing substrate concentrations (Hill coefficient ∼2) (1, 34). The need for fine-tuned conformational changes to elicit an allosteric response by L-Phe is manifested by the loss of positive cooperativity in many PKU-associated hPAH mutants that still maintain the tetrameric structure (19). Current models to explain allosteric regulation with positive cooperative ligand-binding describe the transition from a tense (T), less active state with low affinity for the ligand, to a relaxed (R), more active, high-affinity state. A large conformational transition is indeed visualized for mammalian PAH upon activation by substrate as shown among other by the surfacing of a buried tryptophan residue and an increase in hydrophobicity and size (1, 5, 29, 34). The mechanism of this activation is not yet understood and there is no consensus in the field as to whether the activation is caused by L-Phe binding to an allosteric site in the regulatory domain (22, 35, 36) or to the active site itself (37–39). The numerous studies related to this subject have been recently reviewed by Fitzpatrick (5). The first indication that binding to a regulatory site might be unnecessary to generate positive cooperative response arose when it was shown that noradrenaline, which binds directly to the ferric iron in the active site, also elicited positive cooperativity (37). Later, differential scanning calorimetry investigations further supported that L-Phe only binds to the catalytic domain of hPAH (38). Moreover, surface plasmon resonance analyses and site-directed mutagenesis on crystallographically defined hinges have also identified the active site as the epicenter of the global conformational changes resulting in activation and positive cooperativity observed in the full-length tetrameric enzyme (29). Nevertheless, evidences for L-Phe binding by the isolated regulatory domains have been put forward by isothermal titration calorimetry (22) and nuclear magnetic resonance spectroscopy (36). Interestingly, recent analyses reveal that substrat binding sites in fact exist in the regulatory ACT domain in hPAH, but binding appears obstructed by residues from the catalytic domain (39). It seems therefore not surprising, neither contradictory, that L-Phe binds to isolated ACT domains (22, 36).

The cofactor BH4 also exerts a regulatory inhibitory effect on mammalian PAH, manifested as a stabilization of the T-state, which is reverted by L-Phe activation (1, 5, 27). The negative effect on activity is attributed to the intrinsic autoregulatory sequence (28), and molecular dynamics (MD) simulations complemented with thermodynamic characterizations of BH4-binding also support a restructuring of the intrinsic autoregulatory sequence, which possibly occupies the L-Phe binding site (27). In the liver, PAH exists largely in this BH4-bound, low-activity, stable state (40). The stabilizing effect of BH4 is one of the most important molecular mechanisms behind the stimulation of PKU-mutant activity by BH4 supplementation (i.e., the chaperone effect) (19).

Phosphorylation of mammalian PAH at Ser16 by glucagon-stimulated cAMP-dependent protein kinase increases the affinity for L-Phe and thus activates the enzyme in synergy with L-Phe activation (1, 5). Crystal structures of unphosphorylated and phosphorylated forms of dimeric rat PAH (24) (Table 1) lack structural information on the region around Ser16. Site-directed mutagenesis and molecular modeling indicate that phosphorylation elicits local conformational changes, mostly driven by electrostatics, that increase the accessibility of the substrate binding site (30).

PAH in Non-Mammalian Eukaryote Organisms

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References

The investigation of PAH in non-mammalian organisms including bacteria readily followed the earliest studies of mammalian PAH (41). Recently, PAH has also been identified in some protozoans and slime molds, and even in nonflowering plants (20, 21, 42). While animal PAH uses BH4 as cofactor, other natural tetrahydropterins, such as tetrahydrodictyopterin, are present in D. discoideum together with BH4 and support the PAH function at least in vitro (21, 43). However, BH4 seems to be the PAH cofactor in vivo, while tetrahydrodictyopterin functions mainly as an antioxidant (43). Plant PAH seems to use tetrahydrofolate as cofactor (42).

In addition to their role in the catabolic L-Phe/L-Tyr degradation pathway (7), PAH in lower eukaryotes has been implicated in the synthesis of (pyo)melanin, a well-known pigment that confers advantageous properties (8–10, 20). Melanin is used by insects to encapsulate parasites (9) and by the sponge Geodia cydonium to target cells for apoptosis (8). In the nematode Caenorhabditis elegans, PAH is expressed in the hypodermal cells, and knock-out pah mutants lack a melanin in the cuticle which appears to protect from oxygen-radicals (10).

Bacterial PAH

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References

In bacteria, the gene coding for PAH (phhA) occurs within several phyla, but appears to be relatively scattered within these. The phylogenetic distribution of phhA in bacteria has not been investigated per se, but Pribat et al. report that 184 out of 724 bacterial genomes encode a PAH (44). In some PAH-containing bacteria, phhA is located next to phhB, encoding pterin carbinolamine dehydratase, in the phh operon, which also includes the gene for an aromatic aminotransferase (45). The similarity of the regulatory domain of mammalian PAH with pterin carbinolamine dehydratase has been remarked, and the bacterial operon-structure has been proposed as a possible basis for evolution of the modular gene encoding eukaryotic PAH (24).

Several bacterial PAHs have been studied experimentally to different extents, all showing an absolute requirement for ferrous iron and tetrahydropterin (11, 45–47). It has been speculated that most bacterial PAHs use alternative pterins to BH4, for example, tetrahydromonapterin (44). For the Colwellia psychrerythraea enzyme, highest activity was however measured with BH4 (47). Except for the recently studied PAH from Legionella pneumophila, which appears to be dimeric (11), presently characterized bacterial PAHs are monomeric and show a similar fold to the catalytic domain of mammalian PAH (46, 47) (Table 1; Fig. 2D).

Several pathogens encode a PAH and produce a melanin-pigment, notably a pyomelanin derived from homogentisate autooxidation and polymerization (Fig. 1). The high mutational rate of Pseudomonas aeruginosa during the course of lung infection leads to adaptive mutations in the gene encoding homogentisate oxidase (Fig. 1) (48). This induces increased homogentisate accumulation and hyperproduction of pyomelanin that render the infection very difficult to treat (48). The pyomelanin of the opportunistic pathogen Burkholderia cenocepacia was recently shown to have antioxidant properties, and mutants not producing pyomelanin were more sensitive to oxidative stress (49). Furthermore, the crucial role of L. pneumophila PAH in both the growth of the bacterium in tyrosine-limited media and in the synthesis of pyomelanin has been recently demonstrated (11) (Fig. 3). The secreted pyomelanin has intrinsic ferric reductase activity, contributing to the capacity of Legionella to acquire iron and possibly to its virulence (50).

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Figure 3. Role of PAH in growth and pigment-synthesis in Legionella pneumophila 130b. Wild type 130b and mutant L. pneumophila lacking PAH (NU406) with empty vector (pMMB2002) or vector containing the pah gene (pPhhA) were grown in chemically defined medium (CDM) lacking tyrosine (A) and in medium containing standard (CDM) (B,C) and twice the standard (CDMˆ 2× Tyr) amount of tyrosine (B). The importance of PAH both for growth in media limited in tyrosine (A), and for the production of pyomelanin pigment (B,C) was established. Figure adapted from ref.11. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Adaptive Strategies: Regulation Of PAH Activity By L-Phe

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References

PAH shows an evolutionary adaptation of its structure to accommodate for changes in the regulation of its function. The characterization of PAHs in organisms at different levels of cellular complexity indicates that the tetrameric structure appears early—in an evolutionary scale—in the animal kingdom (21). Clear manifestations of allosteric regulation of PAH activity by L-Phe have only been proven for mammals, but the need for PAH activity in anabolic/catabolic pathways with simultaneous preservation of threshold levels of L-Phe for protein synthesis would require a strict enzyme regulation in all organisms. Several mechanisms appear to contribute to maintain L-Phe homeostasis in different organisms, and, in addition to the regulation by positive cooperativity in mammals, other mechanisms such as non-catalytic binding of L-Phe, and low substrate affinity have been revealed in recent in vitro studies with purified PAHs from C. elegans and bacteria.

PAH from C. elegans, which is tetrameric and includes the canonical three-domain PAH organization and 57% sequence identity with hPAH, does not show positive cooperativity (10). This sophisticated regulatory mechanism that allows mammalian PAH to avoid toxic accumulation of L-Phe is not present in the worm most probably because it is simply not required at its level of nervous system complexity. In fact, knock-out pah worms appear healthy even when grown in media supplemented with excess L-Phe (10), suggesting that accumulation of L-Phe is not detrimental to the C. elegans nervous system.

Nevertheless, it is reasonable to infer that the worm would face the need of maintaining threshold values of L-Phe for protein synthesis. In this context, our discovery of a second binding site for L-Phe in the regulatory domain of worm PAH (39) suggests a simpler regulatory mechanism where the non-catalytic binding of L-Phe, which does not seem to induce activating conformational changes, rather ensures the preservation of certain L-Phe levels.

In the case of bacteria, the cold-adapted PAH from C. psychrerythraea shows a low affinity for L-Phe, and the concentration of substrate providing half maximal activity ([S]0.5, a parameter used in non-Michaelis–Menten kinetics to provide a constant comparable to Km) is 1300 ± 300 μM, much higher than [S]0.5 < 200 μM for eukaryote PAH (1, 10). This could be a result of the increased flexibility and accessibility of the active site due to cold adaptation, but might also indicate a primitive regulatory mechanism to preserve threshold amounts of L-Phe. Indeed, also the thermostable PAH from L. pneumophila displayed low affinity for L-Phe (Km = 735 ± 50 μM). But not all bacterial PAHs seem to have low affinity for its substrate and PAH from C. violaceum has Km (L-Phe) comparable to eukaryote PAH (51). Additional studies are necessary to reveal the extent of low affinity for L-Phe among bacterial PAH. In fact other mechanisms at the transcriptional level, such as the regulation of transcription from phhA by L-Phe shown to take place in Pseudomonas putida (52), might also be important to safeguard L-Phe (and aromatic amino acid) homeostasis.

Evolutionary Change in the Function of the PAH-Catalyzed Reaction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References

It is broadly accepted that the main role of mammalian PAH is the catabolic degradation of dietary L-Phe and protection from HPA. On the other hand, combining the studies from Calvo et al. and Fisher et al. shows that the role of C. elegans PAH is catabolic, but also anabolic, providing L-Phe for melanin synthesis (7, 10). In fact, functional divergence analysis acknowledge that an important functional switch has occurred between the nematode PAH and mammalian PAH on the evolutionary time-scale (39). These results further indicate that the specific residue substitutions are associated to fundamental changes in regulation of the activity (10). The more sophisticated regulation in the mammalian organisms seems to tune the enzyme toward the effective catabolic processing of L-Phe.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References

Authors are very thankful to all members of the Biorecognition research group for discussions, especially to Jarl Underhaug who also contributed to the figures. This research is supported by grants from the Research Council of Norway, the Western Norway Health Authorities, K.G. Jebsen Centre for Research on Neuropsychiatric Disorders and Novo Seeds (Novo Nordisk Fonden).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phenylketonuria
  5. Aromatic Amino Acid Hydroxylases
  6. The Phenylalanine Hydroxylase System
  7. Structure of Mammalian PAH
  8. Regulation of Mammalian PAH by L-Phe, BH4, and Phosphorylation
  9. PAH in Non-Mammalian Eukaryote Organisms
  10. Bacterial PAH
  11. Adaptive Strategies: Regulation Of PAH Activity By L-Phe
  12. Evolutionary Change in the Function of the PAH-Catalyzed Reaction
  13. Acknowledgements
  14. References
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