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Pteridines

Official Journal of the International Society of Pteridinology

Editor-in-Chief: Fuchs, Dietmar


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Volume 25, Issue 2

Issues

Dynamic regulation of phenylalanine hydroxylase

Julian E. Fuchs
  • Corresponding author
  • Institute of General, Inorganic and Theoretical Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, Innsbruck, Austria
  • Unilever Centre for Molecular Science Informatics, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, UK
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/ Dietmar Fuchs
  • Division of Biological Chemistry, Biocenter, Medical University, Innrain 80/82, Innsbruck, Austria
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/ Klaus R. Liedl
  • Institute of General, Inorganic and Theoretical Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, Innsbruck, Austria
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Published Online: 2014-07-23 | DOI: https://doi.org/10.1515/pteridines-2014-0006

Abstract

Phenylalanine hydroxylase (PAH) is the key enzyme in phenylalanine metabolism, catalyzing its oxidative breakdown to tyrosine. Its function in the committed step of amino acid metabolism requires strict regulation. Thus, several regulatory mechanisms are central for an understanding of PAH at the atomistic level. The enzyme is activated by incubation with phenylalanine and inhibited by tetrahydrobiopterin binding. Furthermore, phosphorylation of Ser-16 in the regulatory domain influences enzyme turnover. All major regulatory processes in PAH are connected to the conformational changes within a protein and its oligomeric assembly. The underlying dynamic processes in the enzyme are tackled by a variety of experimental and computational approaches. We especially emphasize the computational approaches, aiming to unravel the changes in the molecular dynamics of PAH that govern allosteric regulation. State-of-the-art molecular dynamics simulations provide access to the conformational transitions in biological macromolecules at the microsecond time scale and beyond. Thus, in silico strategies are promising for the identification of the complex allosteric mechanisms governing PAH activity in vivo.

Keywords: allostery; conformational dynamics; molecular dynamics simulation; phenylalanine hydroxylase; regulation.

Introduction

Phenylalanine hydroxylase (PAH, EC 1.14.16.1) is a key enzyme in human amino acid metabolism and is active in the human liver. PAH catalyses the oxidation of Phe to Tyr at an active site formed by the reductive co-factor (6R)-tetrahydrobiopterin (BH4) and by a non-heme iron in ferrous state. Monooxygenase function is achieved by the incorporation of one oxygen atom from molecular oxygen into the amino acid substrate, while the second atom is bound by the co-factor that forms 4a-hydroxypterin. The underlying catalytic mechanism involving changes in iron oxidation and coordination state is shared by all aromatic acid hydroxylases and has been discussed in detail elsewhere [1].

Dysfunction of PAH leads to the well-studied genetic disease phenylketonuria (PKU), which can be readily detected via phenylketones stemming from the transamination of phenylalanine to phenylpyruvate [2]. Consequently, Phe levels are elevated in PKU patients, leading to neurological disorders [3]. Therefore, a strict diet low in Phe is imposed on PKU patients. The molecular link between elevated Phe levels and neural disorders is not fully clear yet but is believed to involve the amino acid carrier l-type amino acid transporter 1 (LAT1) [4]. Increased Phe concentrations could unbalance the transport of other aromatic amino acids Tyr and Trp, which have a central function as precursors of neurotransmitters. A manifold of mutations has been described in PAH, leading to different forms of PKU. Most mutations lead to PAH misfolding, reduced enzyme stability and impaired molecular motions within a protein [5–7].

Mammalian PAH forms a homotetramer of 50 kDa per subunit. No complete structure of an intact eukaryotic PAH is available to date. Still, large parts can be assembled from different partially overlapping X-ray structures. Each subunit is formed by an N-terminal regulatory domain (residues 1–116) [8], a catalytic domain (residues 117–410) [9], and a short C-terminal coiled coil oligomerization domain (residues 411–452) [10]. The highly flexible residues 1–18, including a phosphorylation site at Ser-16, have not been crystallized yet. Nevertheless, molecular modeling studies identified accessible conformations for this region of PAH [11]. An overview of the complex structural assembly of human tetrameric PAH based on a structural model from Jaffe et al. [12] is shown in Figure 1. The evolutionary aspects of PAH structure and function have recently been reviewed by Flydal and Martinez [13].

Visualizations of the tetrameric state of PAH based on a model of Jaffe et al. [12]: PAH is shown in cartoon representation with the catalytic iron ions as brown spheres. The regulatory domain is colored in red, the catalytic domain in green, and the oligomerization domain in blue. (B) Shows a rotation of (A) by 90° around the y-axis. The red N-terminal regulatory domains restrict the access to the catalytic sites (green).
Figure 1

Visualizations of the tetrameric state of PAH based on a model of Jaffe et al. [12]: PAH is shown in cartoon representation with the catalytic iron ions as brown spheres. The regulatory domain is colored in red, the catalytic domain in green, and the oligomerization domain in blue. (B) Shows a rotation of (A) by 90° around the y-axis. The red N-terminal regulatory domains restrict the access to the catalytic sites (green).

Regulation of PAH activity

The central role of PAH in the committed step of phenylalanine catabolism requires strict regulatory processes. Thus, several molecular mechanisms that regulate enzyme activity at different levels of complexity have evolved.

Activation of PAH by incubation with Phe as well as its inhibition by BH4 has been known for a long time [14]. Phe acts not only as a substrate but also as an activator with positive cooperativity [15]. The binding site of Phe is still controversial. The isolated regulatory domain was shown to bind Phe [16], although there is evidence that Phe also binds directly to the catalytic domain and exhibits regulatory functions [17, 18].

Binding of BH4 does not lead to major changes in the active site of PAH [19]. Still, its binding mode is altered in the presence of an amino acid substrate [20]. BH4 has a stabilizing effect on the three-dimensional structure of PAH, which can be exploited for the design of small molecule pharmacological chaperones [21].

Allosteric regulation of PAH

Besides these presumably active site-directed regulatory effects, several more complex allosteric pathways are described in the literature and have recently been reviewed by Fitzpatrick [22]. Allosteric regulation is a complex phenomenon, where remote sites in an enzyme couple with the active sites. Conformational transitions that occur in a protein emphasize the importance of protein dynamics and involve energy basins in the free-energy landscape of the whole system [23]. Biological macromolecules such as phenylalanine hydroxylase are flexible in solution, thus undergoing steady conformational transitions [24].

A direct link to the allosteric regulation of PAH was shown for PKU-associated PAH mutants. Although maintaining an intact quaternary structure, some mutated enzymes do not exhibit a positive cooperative phenylalanine binding [25]. Thus, the dynamic features in PAH are crucial to rationalize its complex regulation. It is proposed that PAH exists in different states in solution, including a tense (T) and a relaxed (R) state. The tense state exhibits a low binding activity for Phe, whereas the relaxed state represents the high-activity state in the thermodynamic equilibrium. The mechanisms and structures for both states are not clear yet, although it has been shown that the equilibrium of states is also crucial in vivo [26]. Conformational changes between the populated states include the exposure of an additional hydrophobic surface, including Trp-120, and an increase in size upon activation with Phe [27, 28]. Absence of this preactivation with Phe causes a lag phase in Tyr formation due to the high-energy barrier between the two states. Phe binding to the allosteric site was described to be even stronger than active site binding [29]. Phe release from the allosteric site includes major conformational rearrangements and is therefore a slow process [30]. Additionally, an interplay between residues of the catalytic and the regulatory domain has been described [31].

Binding of BH4 induces a population shift in the opposite direction as incubation with Phe. The tense state becomes energetically more favorable, shifting the conformational equilibrium of PAH towards a lower substrate affinity. The dynamic process underlying this shift has been investigated using in silico molecular dynamics simulations, which highlight the diverse dynamic processes in PAH [32]. A rigidification of PAH in the tense state induced by BH4 binding is in agreement with the co-factor’s chaperone activity, stabilizing the protein’s three-dimensional fold [25, 33].

Besides the classical main regulatory mechanisms via Phe and BH4, phosphorylation of Ser-16 was described to activate PAH synergistic to Phe binding [11, 34, 35]. Several kinases were shown to allow phosphorylation at this site, with cAMP-dependent protein kinase A (PKA) as the most likely physiological player [36]. X-ray structures of phosphorylated PAH do not show the phosphorylation site, nor do they show any drastic changes between phosphorylated and unphosphorylated PAH [9]. Thus, molecular modeling provided the only structural insight into the phosphorylation-dependent PAH regulation in solution. Dynamic conformational changes within PAH that render the active site more easily accessible, in agreement with the activating effect of phosphorylation, have been described [11]. The effects of phosphorylation are, in general, smaller compared to Phe-induced transitions [26, 37]. Recently, major changes in hydrogen/deuterium (H/D) exchange kinetics in particular regions of PAH were demonstrated by mass spectrometry [38]. These regions undergo major changes in flexibility and accessibility upon Phe binding, which remained hidden by static X-ray structures.

Further regulatory mechanisms of PAH activity with physiological relevance are the binding of phospholipids (e.g., lysolecithin) [39], modification of Cys-237 [40, 41], possibly oxidative stress [42, 43], as well as several mutations in the protein [44].

A dynamic view of PAH regulation

Recently, a novel paradigm of PAH regulation was proposed. The “morpheein model” holds the changes in oligomeric assembly responsible for altered enzyme activity [45]. Different assemblies of monomers in thermodynamic equilibrium cause differences in the three-dimensional structure of PAH and thus enzymatic turnover. Thus, a high-activity tetramer and a low-activity tetramer of PAH are proposed [34]. The novel morpheein model of PAH is consistent with previously published data on PAH [12]. Additionally, regulation of the quaternary structure level could open interesting new opportunities for drug design [46].

The morpheein model explains the drastic changes in local dynamics upon Phe incubation, as observed by H/D exchange mass spectrometry in a hinge region between the regulatory and the catalytic domain including Trp-120 [47]. This region is also susceptible to limited proteolysis, highlighting its flexibility in the Phe-bound state [48]. With the adaption of the quaternary structure, PAH is mobilized and is, overall, more accessible and flexible. A flexible region near the active site, the Tyr138-loop, is drastically mobilized by Phe incubation and, to a minor extent, by the phosphorylation of Ser-16 [38, 47]. This is in agreement with the activation of the catalytic center, which is otherwise assumed to be inaccessible because of the physical blockage by the autoregulatory N-terminal [9]. Consistently, an N-terminally truncated form of PAH lacking the access restriction to the active site was shown to resemble the activity of the Phe-incubated form [49]. Furthermore, mutations far from the catalytic site of PAH have been shown to influence enzymatic activity. Within the morpheein model, they could shift the conformational equilibrium and thereby affect substrate turnover [50].

The transition between the different morpheeins is believed to include a decomposition of the tetrameric assembly (in fact, a dimer of dimers), which forms intermediate dimers [12]. Thus, major conformational changes are necessary to switch PAH between the states of low and high activity. Methodologies capable of monitoring the underlying dynamic processes within a protein are necessary for a detailed characterization. Molecular dynamics (MD) simulations of biological systems are an interesting alternative to experimental techniques [51, 52], whose originators – Martin Karplus, Michael Levitt, and Arieh Warshel [53] – have been honored recently with a Nobel Prize. In silico simulations allow the tracing of molecular motions in biological systems at atomistic levels at the microsecond time scale [54]. With a dedicated computer infrastructure, simulation trajectories extending over milliseconds have been described [55]. Events accessible to large-scale MD simulations include ligand binding [56], exploration of allosteric communication pathways [57], characterization of local flexibility patterns [58], as well as complete folding simulations of smaller proteins [59, 60].

Several MD studies of PAH have already been reported in the literature. These studies focus on co-factor binding and specificity [32, 61–63], phenylalanine binding to the regulatory domain [64], distinct mutations [44], as well as on the effect of the phosphorylation of Ser-16 [65]. We have recently presented computational studies aiming to characterize the structural effect of oxidative stress via side-chain oxidation [66] and ligand binding to a distal site in a monomeric bacterial PAH [67]. All studies agree that PAH activity critically depends on conformational flexibility and report major changes in enzyme structure and dynamics by the involved effectors. The striking conformational plasticity of PAH is evident from MD-generated structural ensembles, highlighting several mobile domains even within the catalytic domain (see Figure 2A).

(A) Structural ensemble of 10 snapshots extracted from a 200-ns MD simulation of oxidized PAH [66]: The catalytic domain of PAH is shown as a cartoon with the catalytic iron ion as a brown sphere. The X-ray structure is shown in black; accessible structures in the MD ensemble in different shades of green show the diversity of the adopted conformations. Solution dynamics of the highly flexible Tyr-138 can be seen on the top right including the discussed contraction over the active site. (B) Conformational entropy of native and oxidized PAH [66]: Conformational entropy was calculated from 16 parallel replica exchange molecular dynamics simulations for both systems. Entropy values are shown as black bars for the native state and as red bars for the oxidized state. Conformational entropy of the oxidized state exceeds the entropy of the native state at each temperature-equivalent simulation, indicating an unexpected mobilization by the introduction of an additional bond.
Figure 2

(A) Structural ensemble of 10 snapshots extracted from a 200-ns MD simulation of oxidized PAH [66]: The catalytic domain of PAH is shown as a cartoon with the catalytic iron ion as a brown sphere. The X-ray structure is shown in black; accessible structures in the MD ensemble in different shades of green show the diversity of the adopted conformations. Solution dynamics of the highly flexible Tyr-138 can be seen on the top right including the discussed contraction over the active site. (B) Conformational entropy of native and oxidized PAH [66]: Conformational entropy was calculated from 16 parallel replica exchange molecular dynamics simulations for both systems. Entropy values are shown as black bars for the native state and as red bars for the oxidized state. Conformational entropy of the oxidized state exceeds the entropy of the native state at each temperature-equivalent simulation, indicating an unexpected mobilization by the introduction of an additional bond.

Small modifications of the structure of PAH can cause major changes in the system’s dynamics. Reanalysis of the trajectories of native and oxidized PAH from an earlier study [66] indicated unexpected effects. Introduction of an additional disulfide bond to the system does not lead to a rigidification of the system as expected from the elimination of the degrees of freedom but rather to a mobilization and thus to a gain in conformational entropy (see Figure 2B). We quantified the change in conformational entropy by performing a normal mode analysis on the whole trajectories of native and oxidized PAH using the cpptraj program of AmberTools [68]. We included 1000 equal-spaced snapshots of two 100-ns trajectories in our reanalysis after an equilibration phase including 1 ns of unrestrained simulation (see Ref. [66] for details). We calculated the conformational entropy from a quasiharmonic analysis [69] of the mass-weighted covariance matrix of all 16 replica exchange molecular dynamics simulations of both native and oxidized PAH. Besides the expected increase in conformational entropy with increasing temperature, we found the oxidized state of PAH to be more flexible than the native state in all 16 simulation pairs at the same temperature. This trend underlines the described dynamic contraction of the binding site upon side-chain oxidation [66]. Therefore, the dynamic effects in PAH are not easy to predict from static structures. MD simulations are an attractive technique to explore flexibility-related phenomena in PAH in silico.

Based on recent structural models of PAH in native tetrameric assembly, MD studies should be an attractive tool to investigate the changes in enzyme dynamics related to tetramerization and activation state. We believe that the inclusion of these aspects into current computational models would provide a novel insight into the molecular mechanisms of PAH regulation. Enzyme regulation involving changes in assembly state has already been established for several enzymatic systems including influenza neuraminidase. Molecular dynamics simulations were found to be helpful in the characterization of the changes in the system’s dynamics after the formation of additional macromolecular interfaces by tetramerization [70].

Perspectives

Understanding of static structures is insufficient to capture the complex regulatory mechanisms in PAH. Thus, dynamic methods are necessary to allow further insight into the conformational transitions associated with PAH activity and modulation. Conformational dynamics are of crucial importance for a comprehensive picture of biomolecular functions in physiological conditions. Hence, techniques such as MD simulations that allow the tracing of molecular motions at the atomistic level are increasingly important. Once regulatory pathways are sufficiently explored, targeting the respective dynamic hotspots with novel drug candidates could directly lead to the discovery of novel therapeutics for PKU. MD studies can also provide information about the potential modulators of PAH activity that may relate to moderate hyperphenylalaninemia, which often develops in chronic diseases without a PAH-specific genetic background [71]. Thus, protein dynamics could be a key to the structure-based drug design of challenging targets.

Acknowledgments

Julian E. Fuchs thanks the Austrian Academy of Sciences for a DOC fellowship at the Institute of General, Inorganic and Theoretical Chemistry at the University of Innsbruck. The authors thank Aurora Martinez for fruitful discussions and Roland Huber for support in trajectory reanalysis.

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About the article

Corresponding author: Julian E. Fuchs, Institute of General, Inorganic and Theoretical Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, Innsbruck, Austria; and Unilever Centre for Molecular Science Informatics, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, UK, E-mail:


Received: 2014-04-22

Accepted: 2014-05-16

Published Online: 2014-07-23

Published in Print: 2014-07-01


Citation Information: Pteridines, Volume 25, Issue 2, Pages 33–39, ISSN (Online) 2195-4720, ISSN (Print) 0933-4807, DOI: https://doi.org/10.1515/pteridines-2014-0006.

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