Highly efficient production of L-phenylalanine (L-Phe) in E. coli has been achieved by multiple rounds of random mutagenesis and modification of key genes of the shikimate (SHIK) and L-Phe branch pathways. In this study, we performed transcriptomic (16, 24 and 48 h) and metabolomic analyses (8, 16, 24, 32,40, and 48 h) based on time sequences in an engineered E. coli strain producing L-Phe, aiming to reveal the overall changes of metabolic activities during the fermentation process.

Aptamers are ligand-binding RNA or DNA molecules and have been widely examined as biosensors, diagnostic tools, and therapeutic agents. The application of aptamers as biosensors commonly requires an expression platform to produce a signal to report the aptamer-ligand binding event. Traditionally, aptamer selection and expression platform integration are two independent steps and the aptamer selection requires the immobilization of either the aptamer or the ligand. These drawbacks can be easily overcome through the selection of allosteric DNAzymes (aptazymes). Herein, we used the technique of Expression-SELEX developed in our laboratory to select for aptazymes that can be specifically activated by low concentrations of l-phenylalanine. We chose a previous DNA-cleaving DNAzyme known as II-R1 as the expression platform for its low cleavage rate and used stringent selection conditions to drive the selection of high-performance aptazyme candidates. Three aptazymes were chosen for detailed characterization and these DNAzymes were found to exhibit a dissociation constant for l-phenylalanine as low as 4.8 μM, a catalytic rate constant improvement as high as 20 000-fold in the presence of l-phenylalanine, and the ability to discriminate against closely related l-phenylalanine analogs including d-phenylalanine. This work has established the Expression-SELEX as an effective SELEX method to enrich high-quality ligand-responsive aptazymes.

The suite of biological catalysts found in Nature has the potential to contribute immensely to scientific advancements, ranging from industrial biotechnology to innovations in bioenergy and medical intervention. The endeavour to obtain a catalyst of choice is, however, wrought with challenges. Herein we report the design of a structure-based annotation system for the identification of functionally similar enzymes from diverse sequence backgrounds. Focusing on an enzymatic activity with demonstrated synthetic and therapeutic relevance, five new phenylalanine ammonia lyase (PAL) enzymes were discovered and characterised with respect to their potential applications. The variation and novelty of various desirable traits seen in these previously uncharacterised enzymes demonstrates the importance of effective sequence annotation in unlocking the potential diversity that Nature provides in the search for tailored biological tools. This new method has commercial relevance as a strategy for assaying the 'evolvability' of certain enzyme features, thus streamlining and informing protein engineering efforts.

In phenylketonuria (PKU), natural protein tolerance is defined as the maximum natural protein intake maintaining a blood phenylalanine (Phe) concentration within a target therapeutic range. Tolerance is affected by several factors, and it may differ throughout a person's lifespan. Data on lifelong Phe/natural protein tolerance are limited and mostly reported in studies with low subject numbers. This systematic review aimed to investigate how Phe/natural protein tolerance changes from birth to adulthood in well-controlled patients with PKU on a Phe-restricted diet. Five electronic databases were searched for articles published until July 2020. From a total of 1334 results, 37 articles met the eligibility criteria (n = 2464 patients), and 18 were included in the meta-analysis. The mean Phe (mg/day) and natural protein (g/day) intake gradually increased from birth until 6 y (at the age of 6 months, the mean Phe intake was 267 mg/day, and natural protein intake was 5.4 g/day; at the age of 5 y, the mean Phe intake was 377 mg/day, and the natural protein intake was 8.9 g/day). However, an increase in Phe/natural protein tolerance was more apparent at the beginning of late childhood and was >1.5-fold that of the Phe tolerance in early childhood. During the pubertal growth spurt, the mean natural protein/Phe tolerance was approximately three times higher than in the first year of life, reaching a mean Phe intake of 709 mg/day and a mean natural protein intake of 18 g/day. Post adolescence, a pooled analysis could only be performed for natural protein intake. The mean natural protein tolerance reached its highest (32.4 g/day) point at the age of 17 y and remained consistent (31.6 g/day) in adulthood, but limited data were available. The results of the meta-analysis showed that Phe/natural protein tolerance (expressed as mg or g per day) increases with age, particularly at the beginning of puberty, and reaches its highest level at the end of adolescence. This needs to be interpreted with caution as limited data were available in adult patients. There was also a high degree of heterogeneity between studies due to differences in sample size, the severity of PKU, and target therapeutic levels for blood Phe control.

The enzyme phenylalanine ammonia lyase (PAL) controls the transition from primary to secondary metabolism by converting L-phenylalanine (L-Phe) to cinnamic acid. However, the function of PAL in pear plants (Pyrus bretschneideri) has not yet been fully elucidated.

Whether amino acids act on cellular insulin signaling remains unclear, given that increased circulating amino acid levels are associated with the onset of type 2 diabetes (T2D). Here, we report that phenylalanine modifies insulin receptor beta (IRβ) and inactivates insulin signaling and glucose uptake. Mice fed phenylalanine-rich chow or phenylalanine-producing aspartame or overexpressing human phenylalanyl-tRNA synthetase (hFARS) develop insulin resistance and T2D symptoms. Mechanistically, FARS phenylalanylate lysine 1057/1079 of IRβ (F-K1057/1079), inactivating IRβ and preventing insulin from promoting glucose uptake by cells. SIRT1 reverse F-K1057/1079 and counteract the insulin-inactivating effects of hFARS and phenylalanine. F-K1057/1079 and SIRT1 levels in white blood cells from T2D patients are positively and negatively correlated with T2D onset, respectively. Blocking F-K1057/1079 with phenylalaninol sensitizes insulin signaling and relieves T2D symptoms in hFARS-transgenic and db/db mice. These findings shed light on the activation of insulin signaling and T2D progression through inhibition of phenylalanylation.

Phenylketonuria (PKU) is an inherited metabolic disorder caused by mutation within phenylalanine hydroxylase (PAH) gene. Loss-of-function of PAH leads to accumulation of phenylalanine in the blood/body of an untreated patient, which damages the developing brain, causing severe mental retardation. Current gene therapy strategies based on adeno-associated vector (AAV) delivery of PAH gene were effective in male animals but had little long-term effects on blood hyperphenylalaninemia in females. Here, we designed a gene therapy strategy using AAV to deliver a human codon-optimized phenylalanine amino lyase in a liver-specific manner. It was shown that PAL was active in lysing phenylalanine when it was expressed in mammalian cells. We produced a recombinant adeno-associated vector serotype 8 (AAV8) viral vector expressing the humanized PAL under the control of human antitrypsin (hAAT) promoter (AAV8-PAL). A single intravenous administration of AAV8-PAL caused long-term correction of hyperphenylalaninemia in both male and female PKU mice (strain Pahenu2). Besides, no obvious liver injury was observed throughout the treatment process. Thus, our results established that AAV-mediated liver delivery of PAL gene is a promising strategy in the treatment of PKU.

Propensity of phenylalanine (Phe) for nonpolar environments drives its intercalation into phospholipid membranes, which has been implicated in metabolic and neurological disorders. The knowledge of Phe intercalation parameters can be instrumental in understanding various membrane processes triggered by interactions with Phe, in particular the early events leading to the formation of nucleation/docking sites for the self-assembly of Phe amyloid fibrils at the membrane surface.

Phenylketonuria (PKU) is an inborn error of metabolism characterized by pathogenic variants of the phenylalanine hydroxylase (PAH) gene with a resulting accumulation of phenylalanine (Phe) to neurotoxic levels. Diagnosis of PKU in the Latvian population began in 1985 and the present study's aim was to evaluate the available data on all PKU patients in Latvia.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, with its incidence constantly increasing. To date, there is no cure for the disease, with a need for new and effective treatments. Morin hydrate (MH) is a naturally occurring flavonoid of the Moraceae family with antioxidant and anti-inflammatory properties; however, the blood-brain barrier (BBB) prevents this flavonoid from reaching the CNS when aiming to potentially treat AD. Seeking to use the LAT-1 transporter present in the BBB, a nanoparticle (NPs) formulation loaded with MH and functionalized with phenylalanine-phenylalanine dipeptide was developed (NPphe-MH) and compared to non-functionalized NPs (NP-MH). In addition, two formulations were prepared using rhodamine B (Rh-B) as a fluorescent dye (NPphe-Rh and NP-Rh) to study their biodistribution and ability to cross the BBB. Functionalization of PLGA NPs resulted in high encapsulation efficiencies for both MH and Rh-B. Studies conducted in Wistar rats showed that the presence of phenylalanine dipeptide in the NPs modified their biodistribution profiles, making them more attractive for both liver and lungs, whereas non-functionalized NPs were predominantly distributed to the spleen. Formulation NPphe-Rh remained in the brain for at least 2 h after administration.

The rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed.

Phenylalanine hydroxylase deficiency (PAHD), one of the genetic disorders resulting in hyperphenylalaninemia, has a complex phenotype with many variants and genotypes among different populations. Here, we describe the mutational and phenotypic spectrum of PAHD in a cohort of 420 patients from neonatal screening between 1999 and 2016. The observed phenotypes comprised 43.57% classic phenylketonuria, 33.10% mild PKU, and 23.33% mild hyperphenylalaninemia, with an overall PAHD incidence of 1 in 20,445. Genetic testing was performed for 209 patients and 72 variants including seven novel variants were identified. These included two synonymous and five pathogenic nonsynonymous variants (p.S36*, p.T186I, p.L255W, p.F302V and p.R413H). The most common variant among all patients was p.R243Q, followed by p.R241C, p.Y204C, p.R111* and c.442-1G > A. Variants p.R53H and p.F392I occurred only in MHP with 19.3% and 8.0% of the observed alleles respectively. The genotypes p.[R241C];[R243Q], p.[R243Q];[R243Q], and p.[Y204C];[R243Q] were abundant across all PAHD patients. The distributions of the null allele and the three defined genotypes, null/null, null/missense, and missense/missense, were significantly different between the cPKU and mPKU patients. However, no significant differences were found between mPKU and MHP patients, indicating that other modifier factors influence the phenotypic outcome in these patients. The data presented here will provide a valuable tool for improved genetic counseling and management of future cases of PAHD in China.

The inhibition of Plasmodium cytosolic phenylalanine tRNA-synthetase (cFRS) by a novel series of bicyclic azetidines has shown the potential to prevent malaria transmission, provide prophylaxis, and offer single-dose cure in animal models of malaria. To date, however, the molecular basis of Plasmodium cFRS inhibition by bicyclic azetidines has remained unknown. Here, we present structural and biochemical evidence that bicyclic azetidines are competitive inhibitors of L-Phe, one of three substrates required for the cFRS-catalyzed aminoacylation reaction that underpins protein synthesis in the parasite. Critically, our co-crystal structure of a PvcFRS-BRD1389 complex shows that the bicyclic azetidine ligand binds to two distinct sub-sites within the PvcFRS catalytic site. The ligand occupies the L-Phe site along with an auxiliary cavity and traverses past the ATP binding site. Given that BRD1389 recognition residues are conserved amongst apicomplexan FRSs, this work lays a structural framework for the development of drugs against both Plasmodium and related apicomplexans.

Phenylalanine hydroxylase (PAH) deficiency, colloquially known as phenylketonuria (PKU), is among the most common inborn errors of metabolism and in the past decade has become a target for the development of novel therapeutics such as gene therapy. PAH deficient mouse models have been key to new treatment development, but all prior existing models natively express liver PAH polypeptide as inactive or partially active PAH monomers, which complicates the experimental assessment of protein expression following therapeutic gene, mRNA, protein, or cell transfer. The mutant PAH monomers are able to form hetero-tetramers with and inhibit the overall holoenzyme activity of wild type PAH monomers produced from a therapeutic vector. Preclinical therapeutic studies would benefit from a PKU model that completely lacks both PAH activity and protein expression in liver. In this study, we employed CRISPR/Cas9-mediated gene editing in fertilized mouse embryos to generate a novel mouse model that lacks exon 1 of the Pah gene. Mice that are homozygous for the Pah exon 1 deletion are viable, severely hyperphenylalaninemic, accurately replicate phenotypic features of untreated human classical PKU and lack any detectable liver PAH activity or protein. This model of classical PKU is ideal for further development of gene and cell biologics to treat PKU.

The transcriptional regulation of phenylalanine metabolism is particularly important in conifers, long-lived species that use large amounts of carbon in wood. Here, we show that the Pinus pinaster transcription factor, PpNAC1, is a main regulator of phenylalanine biosynthesis and utilization. A phylogenetic analysis classified PpNAC1 in the NST proteins group and was selected for functional characterization. PpNAC1 is predominantly expressed in the secondary xylem and compression wood of adult trees. Silencing of PpNAC1 in P. pinaster results in the alteration of stem vascular radial patterning and the down-regulation of several genes associated with cell wall biogenesis and secondary metabolism. Furthermore, transactivation and EMSA analyses showed that PpNAC1 is able to activate its own expression and PpMyb4 promoter, while PpMyb4 is able to activate PpMyb8, a transcriptional regulator of phenylalanine and lignin biosynthesis in maritime pine. Together, these results suggest that PpNAC1 is a functional ortholog of the ArabidopsisSND1 and NST1 genes and support the idea that key regulators governing secondary cell wall formation could be conserved between gymnosperms and angiosperms. Understanding the molecular switches controlling wood formation is of paramount importance for fundamental tree biology and paves the way for applications in conifer biotechnology.

The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may not be essential for holding the bridging water molecules together, because many of these waters are retained even in the Ala mutant. However, interactions via the bridging waters contribute to cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of phenylalanine. It thus appears that the second-coordination sphere Asp residue in cPAH, and, by extrapolation, the equivalent residue in other AAAHs, plays a role in fine-tuning pterin affinity in the ground state via deformable interactions with bridging waters and assumes a more significant role in the transition state by aligning the cofactor through direct hydrogen bonding.

Nanostructure morphology originating from the self-assembly of molecules has attracted substantial attention due to its role in toxic amyloid fibril formation and immense potential in the design and fabrication of novel biomaterials. This study presents the role of intermolecular electrostatic interaction on the self-assembly process of l-phenylalanine (L-Phe) amino acid. We have employed attenuated total reflection Fourier transform infrared spectroscopy to probe the existence of different ionization states of the amino acid in various pH aqueous solutions. The self-assembly process of L-Phe in the aqueous phase is explored by using circular dichroism absorption and nuclear magnetic resonance spectroscopic tools. The observed spectral features have shown the signature of higher order structures and possible perturbation in the π-π stacking aromatic interactions for the cationic and anionic states of the amino acid. Scanning electron microscopy is used to probe the self-assembled morphology of the L-Phe amino acid dried samples prepared from the same pH aqueous solutions. We find that for the case of zwitterionic states the self-assembly nanostructures are dominated by the presence of fibrillar morphology, however interestingly for cationic and anionic states the morphology is dominated by the presence of flakes. Our finding demonstrates the potential influence of intermolecular electrostatic interaction over the aromatic π-π stacking interaction in hindering the fibril formation.

L-type amino acid transporter 1 (LAT1) transfers essential amino acids across cell membranes. Owing to its predominant expression in the blood-brain barrier and tumor cells, LAT1 has been exploited for drug delivery and targeting to the central nervous system (CNS) and various cancers. Although the interactions of amino acids and their mimicking compounds with LAT1 have been extensively investigated, the specific structural features for an optimal drug scaffold have not yet been determined. Here, we evaluated a series of LAT1-targeted drug-phenylalanine conjugates (ligands) by determining their uptake rates by in vitro studies and investigating their interaction with LAT1 via induced-fit docking. Combining the experimental and computational data, we concluded that although LAT1 can accommodate various types of structures, smaller compounds are preferred. As the ligand size increased, its flexibility became more crucial in determining the compound's transportability and interactions. Compounds with linear or planar structures exhibited reduced uptake; those with rigid lipophilic structures lacked interactions and likely utilized other transport mechanisms for cellular entry. Introducing polar groups between aromatic structures enhanced interactions. Interestingly, compounds with a carbamate bond in the aromatic ring's para-position displayed very good transport efficiencies for the larger compounds. Compared to the ester bond, the corresponding amide bond had superior hydrogen bond acceptor properties and increased interactions. A reverse amide bond was less favorable than a direct amide bond for interactions with LAT1. The present information can be applied broadly to design appropriate CNS or antineoplastic drug candidates with a prodrug strategy and to discover novel LAT1 inhibitors used either as direct or adjuvant cancer therapy.

Selenium (Se)-enriched habitats have led to chronic selenosis, seriously affecting the health and survival of Procapra przewalskii (P. przewalskii). Our targets were to explore the molecular mechanisms of chronic selenosis and to look for a new way to protect endangered species. The mineral contents of soils, grass, blood, and muscle were analyzed. The biochemical indices, antioxidant capability, and immune function were also investigated. The analyses of proteomics and metabolomics were also carried out. The results showed that the Se contents in the muscle and blood of P. przewalskii, and the soil and grass in the Se-enriched habitats were significantly higher than those in healthy pastures. The P. przewalskii in the Se-enriched habitats showed symptoms of anemia, decreased antioxidant capability, and low immune function. A total of 44 differential proteins and 36 differential metabolites were screened by analyzing their proteomics and metabolomics. These differential proteins and metabolites were involved in glycolysis pathway, amino acid biosynthesis, carbon metabolism, phenylalanine metabolism, and energy metabolism. In particular, phenylalanine metabolism was the common pathway of proteomics and metabolomics, which was an important finding in studying the mechanism of chronic selenosis in animals. This study will help us to further understand the mechanism of chronic selenosis in P. przewalskii, and it provides a scientific basis for the protection of endangered species in Se-enriched habitats.

Phytophthora root and stem rot of soybean [Glycine max (L.) Merr.] caused by Phytophthora sojae is a destructive disease worldwide. Phenylalanine ammonia-lyase (PAL) is one of the most extensively studied enzymes related to plant responses to biotic and abiotic stresses. However, the molecular mechanism of PAL in soybean in response to P. sojae is largely unclear. Here, we characterize a novel member of the soybean PAL gene family, GmPAL2.1, which is significantly induced by P. sojae. Overexpression and RNA interference analysis demonstrates that GmPAL2.1 enhances resistance to P. sojae in transgenic soybean plants. In addition, the PAL activity in GmPAL2.1-OX transgenic soybean is significantly higher than that of non-transgenic plants after infection with P. sojae, while that in GmPAL2.1-RNAi soybean plants is lower. Further analyses show that the daidzein, genistein and salicylic acid (SA) levels and the relative content of glyceollins are markedly increased in GmPAL2.1-OX transgenic soybean. Taken together, these results suggest the important role of GmPAL2.1 functioning as a positive regulator in the soybean response to P. sojae infection, possibly by enhancing the content of glyceollins, daidzein, genistein and SA.