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Structural Studies of the Catalytic and Regulatory Mechanisms of Phenylalanine HydroxylaseLi, Jun 2010 August 1900 (has links)
The catalytic and regulatory mechanisms of phenylalanine hydroxylase were
investigated by structural studies of in this research. Phenylalanine hydroxylase (PheH)
hydroxylates phenylalanine to produce tyrosine using tetrahydrobiopterin (BH4) and
oxygen. The three ligands to the iron, His285, His290, and Glu330, were mutated to
glutamine, glutamate, and histidine. All the mutants had low but measurable activity.
Mutation of Glu330 had the greatest effect on activity and mutation of His290 the least.
All the mutations resulted in an excess of tetrahydropterin oxidized relative to tyrosine
formation, with mutation of His285 having the greatest effect on the coupling of the two
partial reactions. All the mutants greatly decreased the affinity for iron, with mutation of
Glu330 the most deleterious. The results complement previous results with tyrosine
hydroxylase in establishing the plasticity of the individual iron ligands in this enzyme
family.
Hydrogen/deuterium exchange and mass spectrometry showed that peptides lying
in the interface between the regulatory and catalytic domains display large increases of
deuterium incorporation in the presence of phenylalanine. However, the effects of phenylalanine on a mutant enzyme lacking the regulatory domain are limited to peptides
surrounding the binding site of phenylalanine. These results support the autoinhibitory
function of the N-terminus of PheH. No peptides show a changed deuterium
incorporation pattern in the presence of BH4, suggesting that BH4 binding does not
change the structure significantly from the resting form. In phosphorylated PheH, three
peptides show a deuterium incorporation pattern similar to that of unphosphorylated
PheH plus phenylalanine, while the other peptides sensitive to phenylalanine binding in
unphosphorylated PheH show the same pattern as that of unphosphorylated PheH
without phenylalanine. Therefore, the conformational changes induced by
phosphorylation are similar to but not identical to those associated with phenylalanine
activation.
The isolated regulatory domain (PheH1-117) was expressed and purified using a QSepharose
column followed by a gel filtration column. Analytical gel filtration shows
that PheH1-117 exists as a dimer in solution. In the presence of phenylalanine, the
retention time of PheH1-117 is significantly changed. The 1H-15N NMR spectra of PheH1-
117 show that the cross-peaks of several residues are altered in the presence of
phenylalanine. These results support the existence of a regulatory binding site for
phenylalanine in the regulatory domain of PheH.
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Pteridine dependent hydroxylases as autoantigens in autoimmune polyendocrine syndrome type 1 /Ekwall, Olov, January 1900 (has links)
Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.
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Mechanistic and Structural Studies of Phenylalanine Hydroxylase from Chromobacterium violaceumPanay Escobar, Aram Joel 2010 August 1900 (has links)
The phenylalanine hydroxylase from Chromobacterium violaceum (CvPheH) is a
non-heme iron monooxygenase that catalyzes the hydroxylation of phenylalanine. This
study presents the use of kinetic isotope effects (KIE) as mechanistic probes to compare
the reactivity of CvPheH and that of the eukaryotic aromatic amino acid hydroxylases.
This study also describes the use of different spectroscopic and kinetic techniques to
identify the hydroxylating intermediate for this enzyme and the assignment of the NMR
backbone resonances of CvPheH.
Kinetic isotope effects on aromatic and benzylic hydroxylation were used to
establish that bacterial and eukaryotic phenylalanine hydroxylases have similar
reactivity. The observed KIE on aromatic hydroxylation of 1.4 was shown to be a
combination of an inverse isotope effect on the hydroxylation of the amino acid and a
normal isotope effect on a subsequent step in the reaction. An isotope effect on benzylic
hydroxylation of 10 was found for CvPheH. This result establishes the similar reactivity
for CvPheH and the eukaryotic aromatic amino acid hydroxylases and suggests the
involvement of a common hydroxylating intermediate.
Kinetic isotope effects were used to study the hydroxylation of the aliphatic
substrate cyclohexylalanine. The Dkcat value with [1,2,2,3,3,4,4,5,5,6,6-2H11]-
cyclohexylalanine is unity with wild-type CvPheH, suggesting that chemistry is not ratelimiting
with this substrate. The intramolecular isotope effect calculated using
[1,2,3,4,5,6-2H6]-cyclohexylalanine yields a value of 14. This result is evidence for the
involvement of a reactive iron species capable of abstracting a hydrogen atom from the
aliphatic carbon in cyclohexylalanine.
Analysis of the CvPheH reaction using freeze-quench Mössbauer spectroscopy
allowed the detection of an Fe(IV) species in the first turnover of the enzyme. Chemical
quench and stopped-flow spectrophotometric methods were used to establish the kinetic
competency of the Fe(IV) intermediate as the hydroxylating species.
The NMR amide backbone resonances in the HSQC spectrum of CvPheH were
assigned to the corresponding amino acid residues using a suite of TROSY-based threedimensional
triple resonance experiments. We were able to assign 224 residues out of
the 278 assignable residues in CvPheH, this constitutes 81 percent of the assignable protein
sequence.
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X-ray characterization of PaPheOH, a bacterial phenylalanine hydroxylaseEkström, Fredrik January 2003 (has links)
<p>Many human diseases are associated with the malfunction of enzymes in the aromatic amino acid hydroxylase family, e.g. phenylketonuria (PKU), hyperphenylalaninemia (HPA), schizophrenia and Parkinson's disease. The family of aromatic aminoacid hydroxylases comprises the enzymes phenylalanine hydroxylase (PheOH), tyrosine hydroxylase (TyrOH) and tryptophane hydroxylase (TrpOH). These enzymes require the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and atomic oxygen. In eukaryotes, the aromatic amino acid hydroxylases share the same organization with a N-terminal regulatory domain, a central catalytic domain and a C-terminal tetramerization domain. Aromatic amino acid hydroxylases that correspond to the core catalytic domain of the eukaryotic enzymes are found in bacteria. The main focus of this thesis is the structural characterization of a phenylalanine hydroxylase from the bacterium Pseudomonas aeruginosa (PaPheOH). </p><p>To initiate the structural characterization, the active site environment was investigated with X-ray absorption spectroscopy (XAS). The experimental data support a model where the active site iron is coordinated by four oxygen atoms and two nitrogen atoms. We suggest that two water molecules, His121, His126 and Glu166 coordinates the active site iron. In this model, Glu166 provides two of the oxygen atoms in a bidentate binding geometry. EXAFS and XANES studies indicate that structural rearrangements are induced in the second and third coordination shells in samples of PaPheOH with BH4 and/or L-Phe. </p><p>The 1.6 Å X-ray structure of PaPheOH shows a catalytic core that is composed of helices and strands in a bowl-like arrangement. The iron is octahedrally coordinated, by two water molecules and the evolutionary conserved His121, His126 and Glu166 that coordinates the iron with bidentate geometry. The pterin binding loop of PaPheOH (residue 81-86) adopts a conformation that is displaced by 5-6 Å from the expected pterin binding site. Consistent with the unfavourable position of the pterin binding loop is the observation that PaPheOH has a low specific activity compared to the enzymes from human and Chromobacterium violaceum. </p><p>The second part of this thesis focus on the crystallization and structure determination of the actin binding domain of a-actinin (ABD). a-Actinin is located in the Z-disc of skeletal muscle were it crosslinks actin filaments to the filamentous protein titin. The ABD domain of a-actinin crystallizes in space group P21 with four molecules in the asymmetric unit. The structure of the ABD domain has been solved to a d-spacing of 2.0 Å. The two CH-domains of ABD is composed of 5 a-helices each. The a-helices fold into a closed compact conformation with extensive intramolecular contacts between the two domains.</p>
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Pteridine dependent hydroxylases as autoantigens in autoimmune polyendocrine syndrome type 1Ekwall, Olov January 2001 (has links)
<p>Autoimmune polyendocrine syndrome type I (APS) is a monogenous, recessively inherited disease characterised by endocrine and non-endocrine autoimmune manifestations. One fifth of APS I patients suffer from periodic intestinal dysfunction with varying degrees of malabsorbtion, steatorrhea and constipation. Alopecia areata is found in one third of APS I patients. By immunoscreening human cDNA libraries derived from normal human duodenum and scalp with APS I sera, we identified tryptophan hydroxylase (TPH) as an intestinal autoantigen and tyrosine hydroxylase (TH) as a dermal autoantigen. Forty-eight percent (38/80) of the APS I patients had TPH antibodies (Ab) and 44% (41/94) showed TH immunoreactivity. No reactivity against TPH or TH was seen in healthy controls. TPH-Abs showed a statistically significant correlation with gastrointestinal dysfunction (p<0.0001) and TH-Abs were significantly correlated to alopecia (p=0.02). TPH-Ab positive APS I sera specifically immunostained TPH containing enterochromaffin cells in normal duodenal mucosa. In affected mucosa a depletion of the TPH containing EC cells was seen. In enzyme inhibition experiments TPH and TH activity <i>in vitro</i> was reduced by adding APS I sera. TPH and TH together with phenylalanine hydroxylase (PAH) constitute the group of pteridine dependent hydroxylases. These are highly homologous enzymes involved in the biosynthesis of neurotransmitters. Immunoprecipitation of PAH expressed <i>in vitro</i> showed that 27% (25/94) of APS I patients had antibodies reacting with PAH, but no associations with clinical manifestations was observed. An immunocompetition assay showed that the PAH reactivity reflects a cross-reactivity with TPH.</p><p>In conclusion, we have identified TPH and TH as intestinal and dermal autoantigens in APS I, coupled to gastrointestinal dysfunction and alopecia. We have also demonstrated immunoreactivity against PAH in APS I patient sera reflecting a cross-reactivity with TPH.</p>
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Pteridine dependent hydroxylases as autoantigens in autoimmune polyendocrine syndrome type 1Ekwall, Olov January 2001 (has links)
Autoimmune polyendocrine syndrome type I (APS) is a monogenous, recessively inherited disease characterised by endocrine and non-endocrine autoimmune manifestations. One fifth of APS I patients suffer from periodic intestinal dysfunction with varying degrees of malabsorbtion, steatorrhea and constipation. Alopecia areata is found in one third of APS I patients. By immunoscreening human cDNA libraries derived from normal human duodenum and scalp with APS I sera, we identified tryptophan hydroxylase (TPH) as an intestinal autoantigen and tyrosine hydroxylase (TH) as a dermal autoantigen. Forty-eight percent (38/80) of the APS I patients had TPH antibodies (Ab) and 44% (41/94) showed TH immunoreactivity. No reactivity against TPH or TH was seen in healthy controls. TPH-Abs showed a statistically significant correlation with gastrointestinal dysfunction (p<0.0001) and TH-Abs were significantly correlated to alopecia (p=0.02). TPH-Ab positive APS I sera specifically immunostained TPH containing enterochromaffin cells in normal duodenal mucosa. In affected mucosa a depletion of the TPH containing EC cells was seen. In enzyme inhibition experiments TPH and TH activity in vitro was reduced by adding APS I sera. TPH and TH together with phenylalanine hydroxylase (PAH) constitute the group of pteridine dependent hydroxylases. These are highly homologous enzymes involved in the biosynthesis of neurotransmitters. Immunoprecipitation of PAH expressed in vitro showed that 27% (25/94) of APS I patients had antibodies reacting with PAH, but no associations with clinical manifestations was observed. An immunocompetition assay showed that the PAH reactivity reflects a cross-reactivity with TPH. In conclusion, we have identified TPH and TH as intestinal and dermal autoantigens in APS I, coupled to gastrointestinal dysfunction and alopecia. We have also demonstrated immunoreactivity against PAH in APS I patient sera reflecting a cross-reactivity with TPH.
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X-ray characterization of PaPheOH, a bacterial phenylalanine hydroxylaseEkström, Fredrik January 2003 (has links)
Many human diseases are associated with the malfunction of enzymes in the aromatic amino acid hydroxylase family, e.g. phenylketonuria (PKU), hyperphenylalaninemia (HPA), schizophrenia and Parkinson's disease. The family of aromatic aminoacid hydroxylases comprises the enzymes phenylalanine hydroxylase (PheOH), tyrosine hydroxylase (TyrOH) and tryptophane hydroxylase (TrpOH). These enzymes require the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and atomic oxygen. In eukaryotes, the aromatic amino acid hydroxylases share the same organization with a N-terminal regulatory domain, a central catalytic domain and a C-terminal tetramerization domain. Aromatic amino acid hydroxylases that correspond to the core catalytic domain of the eukaryotic enzymes are found in bacteria. The main focus of this thesis is the structural characterization of a phenylalanine hydroxylase from the bacterium Pseudomonas aeruginosa (PaPheOH). To initiate the structural characterization, the active site environment was investigated with X-ray absorption spectroscopy (XAS). The experimental data support a model where the active site iron is coordinated by four oxygen atoms and two nitrogen atoms. We suggest that two water molecules, His121, His126 and Glu166 coordinates the active site iron. In this model, Glu166 provides two of the oxygen atoms in a bidentate binding geometry. EXAFS and XANES studies indicate that structural rearrangements are induced in the second and third coordination shells in samples of PaPheOH with BH4 and/or L-Phe. The 1.6 Å X-ray structure of PaPheOH shows a catalytic core that is composed of helices and strands in a bowl-like arrangement. The iron is octahedrally coordinated, by two water molecules and the evolutionary conserved His121, His126 and Glu166 that coordinates the iron with bidentate geometry. The pterin binding loop of PaPheOH (residue 81-86) adopts a conformation that is displaced by 5-6 Å from the expected pterin binding site. Consistent with the unfavourable position of the pterin binding loop is the observation that PaPheOH has a low specific activity compared to the enzymes from human and Chromobacterium violaceum. The second part of this thesis focus on the crystallization and structure determination of the actin binding domain of a-actinin (ABD). a-Actinin is located in the Z-disc of skeletal muscle were it crosslinks actin filaments to the filamentous protein titin. The ABD domain of a-actinin crystallizes in space group P21 with four molecules in the asymmetric unit. The structure of the ABD domain has been solved to a d-spacing of 2.0 Å. The two CH-domains of ABD is composed of 5 a-helices each. The a-helices fold into a closed compact conformation with extensive intramolecular contacts between the two domains.
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Protein Substitute Requirements of Patients with Phenylketonuria on BH4 Treatment: A Systematic Review and Meta-AnalysisIlgaz, Fatma, Marsaux, Cyril, Pinto, Alex, Singh, Rani, Rohde, Carmen, Karabulut, Erdem, Gökmen-Özel, Hülya, Kuhn, Mirjam, MacDonald, Anita 05 May 2023 (has links)
The traditional treatment for phenylketonuria (PKU) is a phenylalanine (Phe)-restricted diet, supplemented with a Phe-free/low-Phe protein substitute. Pharmaceutical treatment with synthetic tetrahydrobiopterin (BH4), an enzyme cofactor, allows a patient subgroup to relax their diet. However, dietary protocols guiding the adjustments of protein equivalent intake from protein substitute with BH4 treatment are lacking. We systematically reviewed protein substitute usage with long-term BH4 therapy. Electronic databases were searched for articles published between January 2000 and March 2020. Eighteen studies (306 PKU patients) were eligible. Meta-analyses demonstrated a significant increase in Phe and natural protein intakes and a significant decrease in protein equivalent intake from protein substitute with cofactor therapy. Protein substitute could be discontinued in 51% of responsive patients, but was still required in 49%, despite improvement in Phe tolerance. Normal growth was maintained, but micronutrient deficiency was observed with BH4 treatment. A systematic protocol to increase natural protein intake while reducing protein substitute dose should be followed to ensure protein and micronutrient requirements are met and sustained. We propose recommendations to guide healthcare professionals when adjusting dietary prescriptions of PKU patients on BH4. Studies investigating new therapeutic options in PKU should systematically collect data on protein substitute and natural protein intakes, as well as other nutritional factors.
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