Structure–Function Relationships in the Aromatic Amino Acid Hydroxylases Enzyme Family: Evolutionary Insights

Skjærven, Lars; Teigen, Knut; Martı́nez, Aurora

doi:10.1002/9780470015902.a0025581

Cited by 5 publications

(11 citation statements)

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“…The aromatic amino acid hydroxylases (AAAHs) are a family of non‐heme iron‐ and tetrahydrobiopterin (BH 4 )‐dependent enzymes that catalyze the hydroxylation of their respective aromatic amino acid substrates using O 2 as an additional substrate (Fitzpatrick, ; Skjærven, Teigen, & Martinez, ). In vertebrates, four genes code for the AAAHs: phenylalanine hydroxylase ( PAH ), tyrosine hydroxylase ( TH ), and tryptophan hydroxylases 1 and 2 ( TPH1 and TPH2 ).…”

Section: Introductionmentioning

confidence: 99%

Phenylalanine hydroxylase variants interact with the co‐chaperone DNAJC12

et al. 2019

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DNAJC12, a type III member of the HSP40/DNAJ family, has been identified as the specific co‐chaperone of phenylalanine hydroxylase (PAH) and the other aromatic amino acid hydroxylases. DNAJ proteins work together with molecular chaperones of the HSP70 family to assist in proper folding and maintenance of intracellular stability of their clients. Autosomal recessive mutations in DNAJC12 were found to reduce PAH levels, leading to hyperphenylalaninemia (HPA) in patients without mutations in PAH. In this work, we investigated the interaction of normal wild‐type DNAJC12 with mutant PAH in cells expressing several PAH variants associated with HPA in humans, as well as in the Enu1/1 mouse model, homozygous for the V106A‐Pah variant, which leads to severe protein instability, accelerated PAH degradation and mild HPA. We found that mutant PAH exhibits increased ubiquitination, instability, and aggregation compared with normal PAH. In mouse liver lysates, we showed that DNAJC12 interacts with monoubiquitin‐tagged PAH. This form represented a major fraction of PAH in the Enu1/1 but was also present in liver of wild‐type PAH mice. Our results support a role of DNAJC12 in the processing of misfolded ubiquitinated PAH by the ubiquitin‐dependent proteasome/autophagy systems and add to the evidence that the DNAJ proteins are important players both for proper folding and degradation of their clients.

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Section: Introductionmentioning

confidence: 99%

Phenylalanine hydroxylase variants interact with the co‐chaperone DNAJC12

et al. 2019

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“…Together with phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH), Tph belongs to the family of pterin-dependent aromatic amino acid hydroxylases (AAAHs), having BH 4 and O 2 as co-substrates and Fe 2+ as cofactor. Crystallographic and mutagenesis studies have shown that AAAHs are characterized by three main functional regions: a regulatory N-terminal region, a catalytic domain, which contains the cofactor and substrate binding sites, and the C-terminal leucine zipper tetramerization domain [ 3 – 5 ]. Despite Tphs have a protein sequence homology of 71%, Tph2 has 44 additional aminoacidic residues at its N-terminus, which are not present in Tph1 [ 1 ].…”

Section: Introductionmentioning

confidence: 99%

Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin

et al. 2015

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Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2 flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2 null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2 null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84–178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2 null/null mice. Finally, we set up a tamoxifen administration protocol that allows an efficient, time-specific inactivation of brain serotonin synthesis. On the whole, we generated a suitable genetic tool to investigate how serotonin depletion impacts on time-specific events during central nervous system development and adulthood life.

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“…TH belongs to the BH 4 -dependent aromatic amino acid hydroxylases (AAAH), a family of enzymes that catalyze physiologically and medically important reactions. Phenylalanine hydroxylase (PAH) and tryptophan hydroxylase 1 and 2 (TPH1 and TPH2) are the rate-limiting enzymes in the degradation of excess L-phenylalanine and the production of serotonin, respectively 3 . The three enzymes are homotetramers, with each subunit consisting of a regulatory ACT domain with an unstructured N-terminal tail of different length, a catalytic domain including the iron coordinated to a 2-His-1-carboxylate facial triad motif 4 , and an oligomerization domain 3 5 .…”

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confidence: 99%

“…Phenylalanine hydroxylase (PAH) and tryptophan hydroxylase 1 and 2 (TPH1 and TPH2) are the rate-limiting enzymes in the degradation of excess L-phenylalanine and the production of serotonin, respectively 3 . The three enzymes are homotetramers, with each subunit consisting of a regulatory ACT domain with an unstructured N-terminal tail of different length, a catalytic domain including the iron coordinated to a 2-His-1-carboxylate facial triad motif 4 , and an oligomerization domain 3 5 . For PAH, structures of the full-length tetramer from rat were recently solved 6 7 but for TH, only truncated forms are available.…”

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confidence: 99%

“…For PAH, structures of the full-length tetramer from rat were recently solved 6 7 but for TH, only truncated forms are available. These have provided the crystal structure of the catalytic and oligomerization domains (PDB IDs 1TOH and 2XSN) 4 and the NMR structure of the regulatory ACT domain (PDB ID 2MDA) 8 and enabled construction of structural models for full-length TH 3 5 8 , which await experimental validation.…”

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confidence: 99%

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Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

Bezem

Baumann

Skjærven

et al. 2016

Sci Rep

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Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners.

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Structure–Function Relationships in the Aromatic Amino Acid Hydroxylases Enzyme Family: Evolutionary Insights

Cited by 5 publications

References 60 publications

Phenylalanine hydroxylase variants interact with the co‐chaperone DNAJC12

Phenylalanine hydroxylase variants interact with the co‐chaperone DNAJC12

Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin

Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

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