Allosteric regulation of phenylalanine hydroxylase

Fitzpatrick, Paul F.

doi:10.1016/j.abb.2011.09.012

Cited by 60 publications

(76 citation statements)

References 104 publications

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“…Thus, Phe allosterically activates PAH by binding to a regulatory domain. Phosphorylation at Ser16 potentiates the effects of Phe, with phosphorylated PAH achieving full activation at lower Phe concentrations than the unphosphorylated protein (5,6). Allosteric activation by Phe is accompanied by a major conformational change, as evidenced by changes in protein fluorescence and proteolytic susceptibility, and by stabilization of a tetrameric conformer (3).…”

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confidence: 99%

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

Arturo

Gupta

Héroux

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

122

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Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phestabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.phenylalanine hydroxylase | phenylketonuria | X-ray crystallography | small-angle X-ray scattering | allosteric regulation M ammalian phenylalanine hydroxylase (PAH) (EC 1.14.16.1) is a multidomain homo-multimeric protein whose dysfunction causes the most common inborn error in amino acid metabolism, phenylketonuria (PKU), and milder forms of hyperphenylalaninemia (OMIM 261600) (1). PAH catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine, using nonheme iron and the cosubstrates tetrahydrobiopterin and molecular oxygen (2, 3). A detailed kinetic mechanism has recently been derived from elegant single-turnover studies (4). PAH activity must be carefully regulated, because although Phe is an essential amino acid, high Phe levels are neurotoxic. Thus, Phe allosterically activates PAH by binding to a regulatory domain. Phosphorylation at Ser16 potentiates the effects of Phe, with phosphorylated PAH achieving full activation at lower Phe concentrations than the unphosphorylated protein (5, 6). Allosteric activation by Phe is accompanied by a major conformational change, as evidenced by changes in protein fluorescence and proteolytic susceptibility, and by stabilization of a tetrameric confo...

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mentioning

confidence: 99%

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

Arturo

Gupta

Héroux

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

122

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“…: 210-567-8264; E-mail: fitzpatrickp@uthscsa.edu. 2 The abbreviations used are: PheH, phenylalanine hydroxylase; BH 4 , tetrahydrobiopterin; TyrH, tyrosine hydroxylase; RDPheH, the regulatory domain of PheH, residues 2-117; RDPheH(25-117), RDPheH residues 25-117; HSQC, heteronuclear single-quantum coherence; PKU, phenylketonuria. …”

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confidence: 99%

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase

Zhang

Fitzpatrick

2016

Journal of Biological Chemistry

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Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25-117) is the regulatory domain of PheH lacking residues 1-24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25-117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25-117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH. Phenylalanine hydroxylase (PheH)2 catalyzes a key step in phenylalanine catabolism, the hydroxylation of phenylalanine to tyrosine in the liver using tetrahydropterin (BH 4 ) and oxygen. A deficiency in human PheH increases the level of phenylalanine in the blood, resulting in the inherited disease phenylketonuria (PKU) (1). Thus, the activity of PheH must be tightly controlled to maintain appropriate phenylalanine levels. PheH is activated by phenylalanine and inhibited by BH 4 (2, 3). In addition, phosphorylation of PheH at Ser-16 is reported to decrease the concentration of phenylalanine required to activate PheH (4).Mammalian PheH is a homotetramer, and each monomer contains an N-terminal regulatory domain, a central catalytic domain, and a C-terminal tetramerization domain. The other two aromatic amino acid hydroxylases, tyrosine hydroxylase (TyrH) and tryptophan hydroxylase, have similar architectures.The crystal structures of the catalytic domains of all three enzymes show very similar folds and active sites (5-7), consistent with these enzymes sharing a common catalytic mechanism (8). The structures of the regulatory domains of PheH and TyrH show that both contain ACT domains (5, 9, 10), although the two enzymes are regulated differently (2, 11). To date, there is no published structure of a full-length mammalian PheH, or indeed of any eukaryotic aromatic amino acid hydroxylase, in that the available structures are of proteins lacking the N-terminal regulatory domain, much of the C-terminal tetramerization domain, or both. The structure of a dimeric form of rat PheH containing both the catalytic and regulatory domains but lacking the C-terminal 24 residues required for tetramer formation (5) has provided the present model for the structural basis for activation by phenylalanine. In this structure the...

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“…The results of this study indicate that the SID Phe : Lys supply can be decreased to 54% without affecting the growth of piglets. This value is much lower than that proposed by current standards (NRC, 1998 and2012;BSAS, 2003). In a cereal-soybean meal-based diet, the Ile, His, and Leu supply may become limiting for growth before Phe and Tyr when the CP content is reduced (van Milgen et al, 2012;Gloaguen et al, 2013).…”

Section: Discussionmentioning

confidence: 55%

“…This means that an equimolar substitution of Tyr by Phe is ineffective to ensure maximum growth. Moreover, the reduction in growth was independent of the SID (Phe + Tyr) level, which indicates that the expected activation of Phe hydroxylase by Phe (Fitzpatrick, 2012) and the subsequent increase in synthesized Tyr from Phe did not lead to higher availability of Tyr for growth. In contrast to our results, Sasse and Baker (1972) determined that Phe was equal in efficacy to Tyr, on a molar basis, to provide Tyr in young chicks.…”

Section: Discussionmentioning

confidence: 88%

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Performance of piglets in response to the standardized ileal digestible phenylalanine and tyrosine supply in low-protein diets

Gloaguen

Floc'H

Primot³

et al. 2014

Animal

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Reducing the CP level of the diet allows for a reduction in N excretion without limiting performance as long as the amino acid (AA) requirements are covered. The availability of crystalline AA has permitted for a considerable reduction in the CP level of diets, practically used in pig nutrition. The adoption of low CP diets and the extent to which the CP content can be reduced further depends on the knowledge of the minimum levels of indispensable AA that maximize growth. The standardized ileal digestible (SID) Phe : Lys and Tyr : Lys requirements and the possibility to substitute Tyr by Phe have never been studied in piglets. The objectives of this study were to estimate these requirements in 10 to 20 kg pig as well as to determine the extent to which Phe can be used to cover the Tyr requirement. In three dose-response studies, six pigs within each of 14 blocks were assigned to six low CP diets (14.5% CP) sub-limiting in Lys at 1.00% SID. In experiment 1, the SID Phe : Lys requirement estimate was assessed by supplementing a Phe-deficient diet with different levels of L-Phe to attain 33%, 39%, 46%, 52%, 58%, and 65% SID Phe : Lys. Because Phe can be used for Tyr synthesis, the diets provided a sufficient Tyr supply. A similar approach was used in experiment 2 with six levels of L-Tyr supplementation to attain 21%, 27%, 33%, 39%, 45% and 52% SID Tyr : Lys. Phenylalanine was supplied at a level sufficient to sustain maximum growth (estimated in experiment 1). The SID Phe : Lys and SID Tyr : Lys requirements for maximizing daily gain were 54% and 40% using a curvilinear-plateau model, respectively. A 10% deficiency in Phe and Tyr reduced daily gain by 3.0% and 0.7%, respectively. In experiment 3, the effect of the equimolar substitution of dietary SID Tyr by Phe to obtain 50%, 57%, and 64% SID Phe : (Phe + Tyr) was studied at two limiting levels of Phe + Tyr. From 57% to 64% SID Phe : (Phe + Tyr), performance was slightly reduced. In conclusion, it is recommended not to use a Phe + Tyr requirement in the ideal AA profile but rather use a SID Phe : Lys of 54% and a SID Tyr : Lys of 40% to support maximal growth.

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Allosteric regulation of phenylalanine hydroxylase

Cited by 60 publications

References 104 publications

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase

Performance of piglets in response to the standardized ileal digestible phenylalanine and tyrosine supply in low-protein diets

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