Phosphorylation of recombinant human phenylalanine hydroxylase: effect on catalytic activity, substrate activation and protection against non-specific cleavage of the fusion protein by restriction protease

Døskeland, Anne P.; Martı́nez, Aurora; Knappskog, Per M.; Flatmark, Torgeir

doi:10.1042/bj3130409

Cited by 62 publications

(96 citation statements)

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“…In agreement with the modeled structure, the formation of an Arg 13 to Ser 16 phosphate salt bridge and the conformational change of the N-terminal tail also explain the higher stability toward limited tryptic proteolysis of the phosphorylated enzyme. The results obtained with the mutant R13A and E381A further support the model proposed for the molecular mechanism for the activation of the enzyme by phosphorylation.…”

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confidence: 61%

Phosphorylation and Mutations of Ser16 in Human Phenylalanine Hydroxylase

Miranda

Teigen

Thórólfsson

et al. 2002

Journal of Biological Chemistry

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Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cyclic AMP-dependent protein kinase is a post-translational modification that increases its basal activity and facilitates its activation by the substrate L-Phe. So far there is no structural information on the flexible N-terminal tail (residues 1-18), including the phosphorylation site. To get further insight into the molecular basis for the effects of phosphorylation on the catalytic efficiency and enzyme stability, molecular modeling was performed using the crystal structure of the recombinant rat enzyme. The most probable conformation and orientation of the N-terminal tail thus obtained indicates that phosphorylation of Ser 16 induces a local conformational change as a result of an electrostatic interaction between the phosphate group and Phenylalanine hydroxylase (PAH 1 ; EC 1.14.16.1, phenylalanine 4-monooxygenase) belongs to the family of aromatic amino acid hydroxylases. PAH catalyzes the hydroxylation of L-Phe to L-Tyr, the rate-limiting step in the catabolism of L-Phe (1, 2), and it requires a nonheme iron, molecular oxygen, and a pterin cofactor for catalysis (3). Genetic defects in the human enzyme (hPAH) cause phenylketonuria, with a broad range of metabolic and clinical phenotypes (4) as well as enzymatic phenotypes (5, 6).hPAH is a tetrameric/dimeric enzyme, and crystal structure analyses (7-9) have shown that each of the chains folds into three domains, i.e. an N-terminal regulatory domain (residues 1-110) that includes the single phosphorylation site Ser 16 , a middle catalytic domain, and a C-terminal oligomerization domain. Mammalian PAH is activated severalfold by preincubation with its substrate L-Phe, which represents the most important mechanism for its regulation in hepatocytes (1). Phosphorylation of PAH at Ser 16 by cAMP-dependent protein kinase (PKA) represents an additional post-transcriptional regulation of the enzyme (10 -14). The two mechanisms of activation are interdependent, i.e. L-Phe enhances its rate of phosphorylation by PKA, and the phosphorylated enzyme requires a lower concentration of substrate for its activation (15,16). It appears that these two mechanisms act synergistically also in vivo and that L-Phe promotes the phosphorylation and activation of PAH in rat liver (13,17). However, the molecular mechanism of this interdependence is not well understood (18,19) and has not been explained by the crystal structure analyses of the phosphorylated (at Ser 16 ) ⌬C24-truncated dimeric form of rat PAH (rPAH) (9). Phosphorylation of the human enzyme results in a mobility shift on SDS-PAGE (20) that is also observed when hPAH is expressed in Escherichia coli (16,21) and in the in vitro transcription-translation system (5, 22). The enzyme expressed in the latter system is recovered as a double band on SDS-PAGE, corresponding to the phosphorylated (ϳ51 kDa) and nonphosphorylated (ϳ50 kDa) forms. Furthermore, we have previously shown by Fourier transform infrared spectroscopy that phosphorylation of the isolated recombi...

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confidence: 61%

Phosphorylation and Mutations of Ser16 in Human Phenylalanine Hydroxylase

Miranda

Teigen

Thórólfsson

et al. 2002

Journal of Biological Chemistry

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“…SDS/PAGE and autoradiography of all the TnT products revealed a double band (Fig. 5 a) with mobilities corresponding to non-phosphorylated (50 kDa) and phosphorylated (51 kDa) recombinant hPAH as previously reported for the E. coli expressed enzyme [4,11,12]. Thus, on further phosphorylation by the catalytic subunit of protein kinase A (PKA) the 50-kDa band disappeared and only the 51-kDa band was observed (Fig.…”

Section: Prokaryotic Expression Of Mutant Forms Of Hpahmentioning

confidence: 85%

“…At timed intervals (0Ϫ 60 min) soybean trypsin inhibitor (in a two-fold superstoichiometric amount to trypsin) was added to 10-µl aliquots of the reaction mixture, and the products of proteolysis were analyzed by SDS/PAGE (see below). Phosphorylation of wt-hPAH (at Ser16) by catalytic subunit of cAMP-dependent protein kinase (PKA) was carried out for 30 min at 30°C [12]. The reaction mixture contained in a total volume of 10 µl: 10 mM potassium phosphate buffer of pH 7.4, 1 mM EDTA, 10 mM magnesium acetate, 1 µg C-subunit of PKA, 200 µM ATP and 3 µl of TnT product.…”

Section: Methodsmentioning

confidence: 99%

Partial characterization and three‐dimensional‐structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria

Bjørgo

Knappskog

Martı́nez

et al. 1998

European Journal of Biochemistry

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The molecular basis for the metabolic defect in patients with phenylketonuria has been characterized for seven missense point mutations (R252G/Q, L255V/S, A259V/T and R270S) and a termination mutation (G272X) in an evolutionarily conserved motif of exon 7 in the catalytic domain of the human phenylalanine hydroxylase (hPAH) gene. The mutations were expressed in three heterologous in vitro systems. When expressed as fusion proteins with maltose-binding protein in Escherichia coli five of the mutant proteins demonstrated a defect in the normal ability of hPAH to fold and assemble as homotetramer/dimer, and they were mostly recovered as inactive aggregated forms. Only for the R252Q and L255V mutants were catalytically active tetramer and dimer recovered and for R252G some dimer, i.e. 20% (R252Q, tetramer), 44% (L255V, tetramer) and 4.4 % (R252G, dimer) of the activity for the respective wild-type (wt) forms. When expressed by a coupled in vitro transcription-translation system, all the mutant enzymes were recovered as a mixture of non-phosphorylated and phosphorylated forms with a low homospecific activity (i.e. maximum 11% of wt-hPAH for the L255V mutant). When transiently expressed in human embryonic kidney (A293) cells a very low level of immunoreactive PAH protein was recovered in spite of normal PAH mRNA levels. All these mutations resulted in variant hPAH proteins which revealed a defect in oligomerization, an increased sensitivity to limited proteolysis in vitro, reduced cellular stability and a variable reduction in their catalytic activity. All these effects seem to result from structural perturbations of the monomer, and based on the crystal structure of the catalytic domain of hPAH, an explanation is provided for the impact of the mutations on the folding and oligomerization of the monomers.Keywords : phenylalanine hydroxylase; mutation; phenylketonuria expression ; protein stability ; threedimensional structure.Hepatic phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase catalyses the hydroxylation of the essential amino acid L-phenylalanine (L-Phe) to form L-tyrosine (L-Tyr) in the presence of tetrahydrobiopterin (H 4 biopterin) and dioxygen. PAH is a soluble cytosolic enzyme which is deficient in phenylketonuria (PKU), an autosomal recessive human disorder, and more than 280 different mutant alleles have been identified in the hPAH gene (for review, see [1] Enzyme. Phenylalanine 3-monooxygenase (EC 1.14.16.1). Note. The coordinates for the crystal structure of the catalytic domain of the dimeric truncated form (residues 103Ϫ452) of wild-type hPAH have the PDB accession code 1PAH. zygous for two different mutations. Our current understanding of the disorder is that the metabolic and clinical phenotypes are mainly determined by the PAH genotype (for review, see [2]).At this point the molecular mechanism of the reduced catalytic activity of mutant hPAH enzymes has been throughly characterized for only a few selected missense mutations, i.e. G46S [3], D143G [4] and Y204C [5]. In the present study...

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“…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).…”

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confidence: 99%

“…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).…”

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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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Phosphorylation of recombinant human phenylalanine hydroxylase: effect on catalytic activity, substrate activation and protection against non-specific cleavage of the fusion protein by restriction protease

Cited by 62 publications

References 35 publications

Phosphorylation and Mutations of Ser16 in Human Phenylalanine Hydroxylase

Phosphorylation and Mutations of Ser16 in Human Phenylalanine Hydroxylase

Partial characterization and three‐dimensional‐structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase

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