Expression of Wild Type and Mutant Forms of Human Phenylalanine Hydroxylase in E. Coli

Author(s):  
Per M. Knappskog ◽  
Hans G. Eiken ◽  
Aurora Martinez ◽  
Sigridur Olafsdottir ◽  
Jan Haavik ◽  
...  
1996 ◽  
Vol 319 (3) ◽  
pp. 941-945 ◽  
Author(s):  
Anne P. DØSKELAND ◽  
Torgeir FLATMARK

Mammalian phenylalanine hydroxylase (PAH) catalyses the conversion of L-phenylalanine to L-tyrosine in the presence of dioxygen and tetrahydrobiopterin; it is a highly regulated enzyme. Little is known about the rates of synthesis and degradation of PAH in vivo. The enzyme has been reported to have a half-life of approx. 2 days in rat liver and 7–8 h in rat hepatoma cells, but the mechanism of its degradation is not known. In the present study it is shown that the tetrameric form of the recombinant wild-type human enzyme is a substrate for the ubiquitin-conjugating enzyme system in the cytosolic fraction of rat testis. Our findings support the conclusion that multi-/poly-ubiquitination of human PAH plays a key role in the turnover of this cytosolic liver enzyme and provides a mechanism for the increased turnover observed for a number of recombinant mutant forms of the enzyme related to the metabolic disorder phenylketonuria, when expressed in eukaryotic cells.


2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Catarina S. Tomé ◽  
Raquel R. Lopes ◽  
Pedro M. F. Sousa ◽  
Mariana P. Amaro ◽  
João Leandro ◽  
...  

An amendment to this paper has been published and can be accessed via a link at the top of the paper.


2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Catarina S. Tomé ◽  
Raquel R. Lopes ◽  
Pedro M. F. Sousa ◽  
Mariana P. Amaro ◽  
João Leandro ◽  
...  

Abstract 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.


2021 ◽  
Vol 13 ◽  
Author(s):  
Abdullah Almotayri ◽  
Jency Thomas ◽  
Mihiri Munasinghe ◽  
Markandeya Jois

Background: The antidepressant mianserin has been shown to extend the lifespan of Caenorhabditis elegans (C. elegans), a well-established model organism used in aging research. The extension of lifespan in C. elegans was shown to be dependent on increased expression of the scaffolding protein (ANK3/unc-44). In contrast, antidepressant use in humans is associated with an increased risk of death. The C. elegans in the laboratory are fed Escherichia coli (E. coli), a diet high in protein and low in carbohydrate, whereas a typical human diet is high in carbohydrates. We hypothesized that dietary carbohydrates might mitigate the lifespan-extension effect of mianserin. Objective: To investigate the effect of glucose added to the diet of C. elegans on the lifespan-extension effect of mianserin. Methods: Wild-type Bristol N2 and ANK3/unc-44 inactivating mutants were cultured on agar plates containing nematode growth medium and fed E. coli. Treatment groups included (C) control, (M50) 50 μM mianserin, (G) 73 mM glucose, and (M50G) 50 μM mianserin and 73 mM glucose. Lifespan was determined by monitoring the worms until they died. Statistical analysis was performed using the Kaplan-Meier version of the log-rank test. Results: Mianserin treatment resulted in a 12% increase in lifespan (P<0.05) of wild-type Bristol N2 worms but reduced lifespan by 6% in ANK3/unc-44 mutants, consistent with previous research. The addition of glucose to the diet reduced the lifespan of both strains of worms and abolished the lifespan-extension by mianserin. Conclusion: The addition of glucose to the diet of C. elegans abolishes the lifespan-extension effects of mianserin.


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