Phenylalanine iminoboronates as new phenylalanine hydroxylase modulators

Montalbano, Francesco; Leandro, João; Farias, Gonçalo D. V.; Lino, Paulo Roque; Guedes, Rita C.; Vicente, João B.; Leandro, Paula; Góis, Pedro M. P.

doi:10.1039/c4ra10306h

Cited by 18 publications

(18 citation statements)

References 24 publications

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“…Enzymatic activity was measured in 100 mM Hepes pH 7.0 in a final reaction volume of 200 µL essentially as described in 39 . The reaction mix was prepared with 5 µg His 6 -hPAH (corresponding to 0.112 µM of tetramer), 1 mM l -Phe (pre-activated condition) and 0.1 mg·mL −1 catalase and incubated for 4 min at 25 °C.…”

Section: Methodsmentioning

confidence: 99%

“…For determination of non-activated hPAH activity, the substrate l -Phe (1 mM) was added simultaneously with BH 4 . After 1 min, the reaction was stopped by adding 200 µL of cold 2% (v/v) acetic acid/ethanol solution, and the amount of produced l -Tyr was quantified by HPLC with fluorescence detection as in 39 . Specific activity is expressed in nmol of l -Tyr produced during 1 min per mg of protein (nmol l -Tyr·min −1 ·mg −1 ).…”

Section: Methodsmentioning

confidence: 99%

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Structure of full-length wild-type human phenylalanine hydroxylase by small angle X-ray scattering reveals substrate-induced conformational stability

Tomé

Lopes

Sousa

et al. 2019

Sci Rep

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Human phenylalanine hydroxylase (hPAH) hydroxylates l-phenylalanine (l-Phe) to l-tyrosine, a precursor for neurotransmitter biosynthesis. Phenylketonuria (PKU), caused by mutations in PAH that impair PAH function, leads to neurological impairment when untreated. Understanding the hPAH structural and regulatory properties is essential to outline PKU pathophysiological mechanisms. Each hPAH monomer comprises an N-terminal regulatory, a central catalytic and a C-terminal oligomerisation domain. to maintain physiological l-Phe levels, hPAH employs complex regulatory mechanisms. Resting PAH adopts an auto-inhibited conformation where regulatory domains block access to the active site. l-phe-mediated allosteric activation induces a repositioning of the regulatory domains. Since a structure of activated wild-type hPAH is lacking, we addressed hPAH l-phe-mediated conformational changes and report the first solution structure of the allosterically activated state. Our solution structures obtained by small-angle X-ray scattering support a tetramer with distorted P222 symmetry, where catalytic and oligomerisation domains form a core from which regulatory domains protrude, positioning themselves close to the active site entrance in the absence of l-phe. Binding of l-Phe induces a large movement and dimerisation of regulatory domains, exposing the active site. Activated hPAH is more resistant to proteolytic cleavage and thermal denaturation, suggesting that the association of regulatory domains stabilises hPAH.The human phenylalanine hydroxylase (hPAH) catalyzes the hydroxylation of l-phenylalanine (l-Phe) into l-tyrosine (l-Tyr). The reaction is the first step in the catabolic pathway of l-Phe/l-Tyr and proceeds to feed neurotransmitter biosynthetic pathways. In non-pathological conditions, degradation of excessive l-Phe by hPAH sustains physiological plasmatic levels of l-Phe (<120 µM) 1 . Deficiency in hPAH leads to phenylketonuria (PKU), characterised by a toxic accumulation of l-Phe and depletion of precursors for neurotransmitter biosynthesis in the central nervous system that overall result in cognitive disability and neurological impairment. Despite being the most prevalent disorder of the amino acid metabolism, the pathophysiology of PKU remains to be fully elucidated and treatment options are mostly limited to a life-long l-Phe-restricted diet 1 . PKU is caused by mutations in the PAH gene, most being missense mutations that affect folding, catalysis and/or regulation of the enzyme 2,3 . Understanding the structural and regulatory properties of hPAH is essential to outline pathophysiological mechanisms in PKU and delineate novel therapeutic strategies. However, the difficult manipulation of recombinant hPAH has thus far hindered its structural characterisation. hPAH is a member of the aromatic

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Structure of full-length wild-type human phenylalanine hydroxylase by small angle X-ray scattering reveals substrate-induced conformational stability

Tomé

Lopes

Sousa

et al. 2019

Sci Rep

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“…Rather than developing a PC molecule directed to the PAH active site, a small molecule that mimics Phe in stabilising the PAH-RD dimerization interface could activate the mutant enzyme above the threshold sufficient for relieving disease phenotypes, and prove useful as an alternative PC therapy, especially for those PKU mutations that are not BH 4 responsive. The recent report of Phe-like molecules that bind PAH could potentially be acting in such a manner 41 , although caution will be needed to avoid cross-reactivity with the active site. Lessons could therefore be learnt from this and other examples of metabolic enzymes (e.g.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

Patel

Kopec

Fitzpatrick

et al. 2016

Sci Rep

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The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme.

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“…The latter runs the risk of constitutive activation (like enzyme replacement therapy), which can deplete Phe levels below those required for normal cellular functions. There have been a variety of approaches to identifying new pharmacological chaperones for PAH, most of which target the enzyme active site (47–50); it is not yet established if these agents selectively bind to and stabilize either RS-PAH or A-PAH.…”

Section: The Pah Structure Equilibrium and Small Molecule Stabilizationmentioning

confidence: 99%

New protein structures provide an updated understanding of phenylketonuria

Jaffe

2017

Molecular Genetics and Metabolism

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Phenylketonuria (PKU) and less severe hyperphenylalaninemia (HPA) constitute the most common inborn error of amino acid metabolism, and is most often caused by defects in phenylalanine hydroxylase (PAH) function resulting in accumulation of Phe to neurotoxic levels. Despite the success of dietary intervention in preventing permanent neurological damage, individuals living with PKU clamor for additional non-dietary therapies. The bulk of disease-associated mutations are PAH missense variants, which occur throughout the entire 452 amino acid human PAH protein. While some disease-associated mutations affect protein structure (e.g. truncations) and others encode catalytically dead variants, most have been viewed as defective in protein folding/stability. Here we refine this view to address how PKU-associated missense variants can perturb the equilibrium among alternate native PAH structures (resting-state PAH and activated PAH), thus shifting the tipping point of this equilibrium to a neurotoxic Phe concentration. This refined view of PKU introduces opportunities for the design or discovery of therapeutic pharmacological chaperones that can help restore the tipping point to healthy Phe levels and how such a therapeutic might work with or without the inhibitory pharmacological chaperone BH4. Dysregulation of an equilibrium of architecturally distinct native PAH structures departs from the concept of “misfolding”, provides an updated understanding of PKU, and presents an enhanced foundation for understanding genotype/phenotype relationships.

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Phenylalanine iminoboronates as new phenylalanine hydroxylase modulators

Abstract: Herein we report the discovery of new modulators of human phenylalanine hydroxylase (hPAH) inspired by the structure of its substrate and regulator l-phenylalanine.

Cited by 18 publications

References 24 publications

Structure of full-length wild-type human phenylalanine hydroxylase by small angle X-ray scattering reveals substrate-induced conformational stability

Structure of full-length wild-type human phenylalanine hydroxylase by small angle X-ray scattering reveals substrate-induced conformational stability

Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

New protein structures provide an updated understanding of phenylketonuria

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