Studies toward the mechanism of allosteric activation in phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH, EC: 1.14.16.1) is a non-heme iron tetrahydropterin-dependent monooxygenase that maintains phenylalanine (L-Phe) homeostasis via conversion of L-Phe to L-Tyr. PAH is an allosteric enzyme that converts from an inactive T-state to an active R-state upon addition of substrate, L-Phe. Allosteric activation is correlated with physical and structural changes within the enzyme and a large activation energy. Crystal structures of PAH have not identified the location of the allosteric effector binding site. Herein, we report computational protein mapping efforts using the FTmap algorithm and experimental site-directed mutagenesis studies designed to define and screen possible L-Phe allosteric binding sites. Mass spectroscopic analysis of PAH proteolytic fragments obtained after photo-crosslinking with 2-azido-3-phenylpropanoate overlapped with one computationally derived allosteric binding pocket containing residues 110-120 and 312-317. Ligand docking studies, fluorescence measurements, binding affinity and activity assays on wild-type and mutant enzymes further characterized the shape and specificity of this pocket. Thermodynamic studies using surface acoustic wave (SAW) biosensing determined the affinity of L-Phe for the allosteric site. Two L-Phe binding sites were observed upon SAW titrations, corresponding to the active and allosteric sites respectively ( K D,app^on 113 ± 12 µM active site, K D,app^on 680 ± 20 µM allosteric site). Site-directed mutagenesis was performed to prepare mutant enzymes containing a single tryptophan (L-Trp) residue. The fluorescence signatures of each of the three native L-Trp residues in PAH were determined by titrations with L-Phe. Trp187 primarily reports L-Phe induced allosteric conformational changes, while Trp120 reports active site L-Phe binding. Trp326 reports small signals of both active and allosteric site changes. Variable temperature stopped-flow fluorescence kinetic studies elucidated a working mechanism for L-Phe allosteric activation of PAH. Fluorescent signals from wild-type, single, and double L-Trp PAH mutants have been used to build kinetic mechanisms for the L-Phe binding in each subunit and subsequent active site reorganization or allosteric conformational change. In these mechanisms, the enzyme has reduced activity (1-2% of wtPAH) until both L-Phe induced active and allosteric site conformational changes have occurred. Failure of either activation step prevents enzyme turnover and is the chemical-based cause of the metabolic condition phenylketonuria

Aromatic amino acid requirement for pregnant swine

Two trials were conducted to determine the total aromatic amino acids (TAAA) and phenylalanine (L-PHE) requirement for pregnant swine. In trial one, twelve Yorkshire x Landrace (Y x L) gravid gilts and in trial two six Y x L gravid gilts were assigned to 6 x 6 Latin-square experiments from d 40 to d 100 of gestation. A basal 12% protein diet of dextrose, corn starch, dried whey, L-glutamic acid, solka floc, soybean oil, amino acids, minerals and vitamins was fed at a rate of 1.82 kg/d. In trial one, the basal diet (.13% L-PHE plus L-TYR) was supplemented with L-PHE to provide 2.37 (basal), 4.19, 6.00, 7.83, 9.65, and 11.47 grams/day (g/d) TAAA. In trial two, the basal diet was supplemented with 6.00 g/d L-TYR and 1.46 (basal), 2.27, 3.27, 4.20, 5.10, and 6.00 g/d L-PHE. Each treatment diet was fed within each of six 10-d periods. In trial one, urine nitrogen (N) excretion decreased, while daily (N) retention increased (lin. P \u3c .01, quad. P \u3c .05) with increasing levels of TAAA intake to a breakpoint of .43% and .42% or 7.38 and 7.64 g/d TAAA, respectively. Fasting and postfed plasma urine N concentrations decreased (lin. and quad. P \u3c .01) to a breakpoint of .37% and .34% or 6.73 and 6.19 g/d, respectively. Urine urea decreased (lin. P \u3c .01) with increasing dietary TAAA to 9.65 g/d or .53% intake. Plasma L-PHE and L-TYR increased (lin. P \u3c .01) as dietary total TAAA intake increased. In trial two, maximum N retention and minimum urine N excretion (lin. P \u3c .05) occurred when 5.10 g/d L-PHE (.28% of diet) were consumed. Plasma urea N did not change significantly, however, the lowest level occurred at an intake of 2.73 g/d L-PHE (.15% of diet). Urine N responded (quartic, P \u3c .05) to increasing levels of L-PHE with minimum excretion occurring when 4.2 g/d L-PHE (.23% of diet) were consumed. As L-PHE intake increased plasma L-PHE concentrations postfed increased (lin. P \u3c .01) and 3.27 g/d L-PHE (.18% of diet) increased postfed concentrations equal to fasted concentrations. Fasting levels of L-TYR increased (lin. P \u3c .05; cubic, P \u3c .01) with increasing levels of L-PHE intake while postfed concentration did not change. Considering the criteria evaluated in trial 1 and adjusting for diets containing natural feedstuffs, .41% or 7.44 g/d TAAA seems to meet the dietary requirement for pregnancy. Response criteria in trial 2 suggest a minimal L-PHE intake of .24% or 4.35 g/d

Three protected tetrapeptides

The structures of three protected tetrapeptides, containing the Boc-Gly-Gly-Phe-X-OMe chain, tert-butoxycarbonyl-glycy-glycl-phenylalanine-leucine methyl ester dihydrate, Boc-Gly-Gly-L-Phe-D-Leu-OMe, C25H38N4O7·2H2O, tert-butoxycarbonyl-glycy-glycl-phenylalanine-methionine methyl ester dihydrate, Boc-Gly Gly-L-Phe-D-Met-OMe, C24H36N4O7S.2H2O and tert-butoxycarbonyl-glycy-glycl-phenylalanine-norleucine methyl ester dihydrate, Boc-Gly-Gly-D-Phe-L-Nle-OMe, C25H38N4O7.2H2O, are described. The three molecules have the same conformation of the Boc-Gly Gly Phe-X-OMe tetrapeptide chain and display the same packing, consisting of couples of molecules linked head-to-tail by two hydrogen (N-HO) bonds; other hydrogen bonds, also involving two water molecules of crystallization, link these couples together, and give rise to a planar structure

Heterochirality Restricts the Self-Assembly of Phenylalanine Dipeptides Capped with Highly Aromatic Groups

The influence of stereochemistry on the self-assembly of phenylalanine (Phe) dipeptides bearing aromatic fluorenyl groups at both the N- and C-termini (Fmoc, OFm) has been investigated. For this purpose, Fmoc–d-Phe–l-Phe–OFm and Fmoc–l-Phe–l-Phe–OFm have been examined considering a wide variety of solvents, which differ in dielectric constant and volatility. Results reveal that replacement of l-Phe by d-Phe has a major impact on the self-assembly propensities, restricting drastically the structural diversity and polymorphism shown by the homochiral dipeptide. Thus, the analogous heterochiral dipeptide shows a great propensity to form micro/nanofibers, independently of the environmental conditions. Theoretical calculations revealed that the stability of antiparallel disposition is much higher (a factor of ca. 15) for Fmoc–d-Phe–l-Phe–OFm than that for Fmoc–l-Phe–l-Phe–OFm, which has been attributed to the hydrophobic core formed in the former. Overall, results suggest that control of the backbone chirality is a potent and versatile strategy to drive and finely tune the self-assembly propensities of highly aromatic peptides.This work was supported by MINECO (RTI2018-098951-B-I00, RTI2018-101827-B-I00, and CTQ2013-40855-R), AGAUR (2017SGR359 and 2017SGR373), and Gobierno de Aragon (research group Aminoacidos y Peptidos E19_20R). Support for the research of C.A. was received through the prize “ICREA Academia” for excellence in research funded by the Generalitat de Catalunya

Structure of full-length wild-type human phenylalanine hydroxylase by small angle X-ray scattering reveals substrate-induced conformational stability

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.publishe

Tracking amino acid’s uptake into the protozoan Acanthamoeba castellanii by stable-isotope labelling and Raman spectral imaging

The capacity of pathogens to acquire nutrients from their host cells is one of the most fundamental aspects of infection biology. Hence, measuring the patterns of nutrients’ uptake by pathogens is essential for understanding the interactions of pathogens with eukaryotic host cells. In this study, we optimized a technique that allows fast and non-destructive measurement of the amino acid Phenylalanine (Phe) acquired by the trophozoite stage of the protozoan Acanthamoeba castellanii (A. castellanii) as they engage with individual human retinal pigment epithelial cells (ARPE-19). ARPE-19 host cells were pre-saturated with Deuterated Phe (L-Phe(D8)) to replace the native substrate Phe (L-Phe). The uptake of L-Phe(D8) by A. castellanii trophozoites was measured by Raman microspectroscopy. This approach allowed us to characterize the uptake patterns of this essential amino acid into A. castellanii trophozoites at a single cell level. At 24 hours post infection (PI) A. castellanii trophozoites are capable of salvaging L-Phe(D8) from host cells. The uptake pattern was time-dependent during the first 24 hours of infection and complete substitution with L-Phe(D8) in all parasites was detected at 48 hours PI. On the other hand, isolated A. castellanii trachyzoites (grown without host cells) did not show significant uptake for L-Phe(D8) from the media; only achieved an uptake ratio of 16-18% of L-Phe(D8) from the culture medium after 24 hours. These findings demonstrate the potential of combining Raman microspectroscopy and stable isotope labelling approaches to elucidate the role of metabolism in mediating A. castellanii interaction with host cells

Tracing amino acid exchange during host-pathogen interaction by combined stable-isotope time-resolved Raman spectral imaging

This study investigates the temporal and spatial interchange of the aromatic amino acid phenylalanine (Phe) between human retinal pigment epithelial cell line (ARPE-19) and tachyzoites of the apicomplexan protozoan parasite Toxoplasma gondii (T. gondii). Stable isotope labelling by amino acids in cell culture (SILAC) is combined with Raman micro-spectroscopy to selectively monitor the incorporation of deuterium-labelled Phe into proteins in individual live tachyzoites. Our results show a very rapid uptake of L-Phe(D8) by the intracellular growing parasite. T. gondii tachyzoites are capable of extracting L-Phe(D8) from host cells as soon as it invades the cell. L-Phe(D8) from the host cell completely replaces the L-Phe within T. gondii tachyzoites 7–9 hours after infection. A quantitative model based on Raman spectra allowed an estimation of the exchange rate of Phe as 0.5–1.6 × 104 molecules/s. On the other hand, extracellular tachyzoites were not able to consume L-Phe(D8) after 24 hours of infection. These findings further our understanding of the amino acid trafficking between host cells and this strictly intracellular parasite. In particular, this study highlights new aspects of the metabolism of amino acid Phe operative during the interaction between T. gondii and its host cell

Role of Ca2+ and L-Phe in Regulating Functional Cooperativity of Disease- Associated ‘‘Toggle’’ Calcium-Sensing Receptor Mutations

The Ca2+-sensing receptor (CaSR) regulates Ca2+ homeostasis in the body by monitoring extracellular levels of Ca2+ ([Ca2+]o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca2+]o–induced [Ca2+]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca2+]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-offunction CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca2+]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu173, which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro221 and Leu173 are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding