Conversion Of Phenylalanine Research Articles

Monitoring of Phenylalanine Levels in Patients with Phenylketonuria Using Dried Blood Spots: a Comparison of Two Methods

Abstract Phenylketonuria (PKU) is caused by deficient activity of phenylalanine hydroxylase (PAH), responsible for the conversion of phenylalanine (Phe) to tyrosine (Tyr). Monitoring of patients with PKU requires the measurement of Phe in plasma using high-performance liquid chromatography (HPLC) or in dried blood spots (DBS) using different techniques to adjust treatment strategy. The objective of this study was to evaluate Phe levels in DBS measured by two different methods and compare them with Phe levels measured in plasma by HPLC. We analyzed 89 blood samples from 47 PKU patients by two different methods: fluorometric method developed in-house (method A) and the commercially available PerkinElmer® Neonatal Phenylalanine Kit (method B) and in plasma by HPLC. The mean Phe levels by method A, method B, and HPLC were 430.4±39.9μmol/L, 439.3±35.4μmol/L, and 442.2±41.6μmol/L, respectively. The correlation values between HPLC and methods A and B were 0.990 and 0.974, respectively (p < 0.001 for both). Our data suggest that methods A and B are useful alternatives for monitoring Phe levels in patients with PKU, with method A being in closer agreement with the reference standard (HPLC).

Screening of MYH7 gene mutation sites in hypertrophic cardiomyopathy and its significance.

Background:There have been few reports of mutations in the beta-myosin heavy chain (MYH7) gene in hypertrophic cardiomyopathy (HCM), which is associated with sudden cardiac death caused by HCM. This study aimed to screen the mutation sites in the sarcomeric gene MYH7 in Chinese patients with HCM. We also planned to analyze the pathogenicity of the mutation site as well as its significance in clinical and forensic medicine.Methods:From January 2006 to June 2017, autopsy cases were collected from the Department of Pathology, the Affiliated Hospital of Qingdao University. The experiment was to detect MYH7 gene status in formalin-fixed paraffin-embedded tissues from 18 independent autopsy cases who suffered HCM related sudden death (fatal HCM) and 20 cases without cardiomyopathy. Common mutation exon fragments of MYH7 gene were amplified by polymerase chain reaction. The end-of-deoxygenation method and gene cloning method were further performed to analyze the mutation sites. Homologous comparison among mutant sites was conducted using BLAST online database.Results:The 1336th nucleotide of MYH7 gene at exon 14 was converted from T to G in one HCM case, resulting in the conversion of threonine (Thr) at position 446 to proline (Pro). In another case, the 1402th nucleotide at exon 14 was converted from T to C, resulting in the conversion of phenylalanine (Phe) at position 468 to leucine (Leu). Homologous comparison results showed that the two amino acid residues of Thr446 and Phe468 are highly conserved among different species.Conclusions:Our results showed fatal HCM harbored mutations of Thr446Pro and Phe468Leu in the MYH7 gene. It is significant for clinical and forensic medicine to further explore the functions and detailed mechanisms of these mutations.

Phenylacetaldehyde synthase 2 does not contribute to the constitutive formation of 2-phenylethyl-β-D-glucopyranoside in poplar

ABSTRACTIn response to herbivory, poplar produces among other compounds the volatile alcohol 2-phenylethanol and its corresponding glucoside 2-phenylethyl-β-D-glucopyranoside. While the free alcohol is released only upon herbivory, the glucoside accumulates also in undamaged leaves, but increases after herbivore feeding. Recently we showed that 2-phenylethanol and its glucoside are biosynthesized via separate pathways in Populus trichocarpa. The phenylacetaldehyde synthase PtAAS1 plays a central role in the de novo formation of herbivory-induced volatile 2-phenylethanol, while the phenylalanine decarboxylase PtAADC1 initiates a pathway responsible for the herbivory-induced production of 2-phenylethyl-β-D-glucopyranoside. Besides PtAAS1, P. trichocarpa possesses another aromatic aldehyde synthase PtAAS2 with in vitro enzymatic activity comparable to that of PtAAS1. However, in contrast to PtAAS1, which is exclusively expressed in herbivory-damaged leaves, PtAAS2 was found to be expressed at constant levels in both damaged and undamaged leaves. Thus it has been hypothesized that PtAAS2 provides phenylacetaldehyde as substrate for the constitutive formation of 2-phenylethyl-β-D-glucopyranoside in undamaged P. trichocarpa trees. By generating RNAi-mediated AAS2 knockdown plants, we show here that despite the similar activities of PtAAS1 and PtAAS2 in vitro, the latter enzyme does not contribute to the biosynthesis of 2-phenylethyl-β-D-glucopyranoside in planta. Based on the recent finding that phenylpyruvic acid accumulates in undamaged poplar leaves, the constitutive formation of the glucoside may now be suggested to proceed via the Ehrlich pathway, which begins with the conversion of phenylalanine into phenylpyruvic acid.

Development and validation of breeder-friendly functional markers of sugary1 gene encoding starch-debranching enzyme affecting kernel sweetness in maize (Zea mays)

The sweet corn variety of maize (Zea mays L.) has become popular worldwide. The recessive allele of sugary1 (su1) encoding starch de-branching enzyme has been much used for sweet corn cultivar development. Here, we aimed to develop su1-based functional marker(s) by using six diverse inbred lines of sugary type and five inbred lines of wild type, and using 27 overlapping primers. In total, 12 indels (insertion and deletion) and 96 SNPs (single nucleotide polymorphisms) were identified that clearly differentiated the dominant and recessive allele of su1. Among these, a 36-bp indel (at position 1247) in the promoter region included a TATA-box, and a 6-bp indel (at position 6456) in intron-10 was predicted to have SRp40 exon-splicing enhancer. Nucleotide substitution in exon-2 at position 2703 (SNP-2703) was involved in C to G mutation leading to conversion of phenylalanine to leucine. The 6-bp and 36-bp indels and SNP-2703 were used to develop breeder-friendly codominant markers: SuDel6-FR, SuDel36-FR and SNP2703-CG-85/89. All three markers were validated in five F2 populations, and SuDel36-FR and SNP2703-CG-85/89 were validated in a set of 230 diverse inbreds having both mutant and wild-type alleles of Su1. This is the first report of development and validation of universal functional markers for su1. These markers (SuDel36-FR and SNP2703-CG-85/89) assume great significance in marker-assisted breeding program.

Synergistic Effect of a Thermal Reaction and Vacuum Dehydration on Improving Xylose-Phenylalanine Conversion to N-(1-Deoxy-d-xylulos-1-yl)-phenylalanine during an Aqueous Maillard Reaction.

The synergistic effect of a thermal reaction and vacuum dehydration on the conversion of xylose (Xyl) and phenylalanine (Phe) to a Maillard-reaction intermediate (MRI) was researched. The yield of N-(1-deoxy-α-d-xylulos-1-yl)-phenylalanine was successfully improved and increased from 13.62 to 47.23% through the method combining a thermal reaction and vacuum dehydration. A dynamic process was involved in the transformation of Xyl and Phe (Xyl-Phe) to N-substituted d-xylosamine and in the transformation of N-substituted d-xylosamine to N-(1-deoxy-α-d-xylulos-1-yl)-phenylalanine during the initial stage of dehydration; then, only the transformation of N-substituted d-xylosamine to N-(1-deoxy-α-d-xylulos-1-yl)-phenylalanine occurred during the final stage. Furthermore, the MRI was prepared under optimized conditions (90 °C and pH 7.4), and the obtained MRI was characterized and confirmed by ESI mass spectrometry and NMR.

Transcriptome sequencing and analysis during seed growth and development in Euryale ferox Salisb

BackgroundEuryale ferox Salisb., an annual aquatic plant, is the only species in the genus Euryale in the Nymphaeaceae. Seeds of E. ferox are a nutritious food and also used in traditional Chinese medicine (Qian Shi in Mandarin). The molecular events that occurred during seed development in E. ferox have not yet been characterized. In this study, we performed transcriptomic analysis of four developmental stages (T1, T2, T3, and T4) in E. ferox seeds with three biological replicates per developmental stage to understand the physiological and biochemical processes during E. ferox seeds development.Results313,844,425 clean reads were assembled into 160,107 transcripts and 85,006 unigenes with N50 lengths of 2052 bp and 1399 bp, respectively. The unigenes were annotated using five public databases (NR, COG, Swiss-Prot, KEGG, and GO). In the KEGG database, all of the unigenes were assigned to 127 pathways, of which phenylpropanoid biosynthesis was associated with the synthesis of secondary metabolites during E. ferox seed growth and development. Phenylalanine ammonia-lyase (PAL) as the first key enzyme catalyzed the conversion of phenylalanine to trans-cinnamic acid, then was related to the synthesis of flavonoids, lignins and alkaloid. The expression of PAL1 reached its peak at T3 stage, followed by a slight decrease at T4 stage. Cytochrome P450 (P450), encoded by CYP84A1 (which also called ferulate-5-hydroxylase (F5H) in Arabidopsis), was mainly involved in the biosynthesis of lignins.ConclusionsOur study provides a transcriptomic analysis to better understand the morphological changes and the accumulation of medicinal components during E. ferox seed development. The increasing expression of PAL and P450 encoded genes in phenylpropanoid biosynthesis may promote the maturation of E. ferox seed including size, color, hardness and accumulation of medicinal components.

Regulation of Tetrahydrobiopterin (BH4) Synthesis in the Nematode C. elegans

5,6,7,8‐tetrahydrobiopterin (BH4) is a cofactor required by the aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases, and alkylglycerol monooxygenase (Werner et al., 2011, Biochem. J. 438: 397). Among the AAAHs are the rate‐limiting synthetic enzymes for serotonin and dopamine synthesis, and for conversion of phenylalanine (Phe) to tyrosine. In humans, the activity of phenylalanine hydroxylase (PAH) is essential for Phe catabolism; dysfunction of PAH, either from mutations in the PAH gene or in genes required for BH4 synthesis, results in phenylketonuria. Regulation of BH4 levels is important for many other physiological functions. The first and rate‐limiting step in BH4 de novo synthesis is performed by GTP Cyclohydrolase I (GTPCH1). Like in many biosynthesis pathways, this first enzyme is regulated by ‘end product feedback inhibition’ whereby BH4 inhibits GTPCH1 activity. In mammals, BH4 does not bind to GTPCH1 alone, but requires an additional protein called GTPCH1 feedback regulatory protein (GFRP) to inhibit GTPCH1. Crystal structures of rat GTPCH1 and GFRP with the BH4 analog BH2 show that the biopterin binds at the interface between the proteins (Maita et al., 2004, J Biol Chem 279: 51534). Phe increases BH4 production via GTPCH1 stimulation, also in a GFRP‐dependent fashion, similarly binding at the interface between GTPCH1 and GFRP. We have recently described the function and expression of genes required for BH4 synthesis and regeneration in the nematode C. elegans (Loer et al., 2015, Genetics 200: 237). Here we present work on the function of the gene gfrp‐1, which encodes a ortholog of mammalian GFRP, in the regulation of BH4 synthesis in C. elegans. Sequence analysis and structural predictions suggest the worm GFRP will function like the mammalian protein, binding both BH4 and Phe. In mammals, GTPCH1 and GFRP can also bind the selective inhibitor 2,4‐Diamino‐6‐hydroxypyrimidine (DAHP), which is structurally similar to both GTP (substrate) and BH4 (pathway end product). At low concentrations, DAHP apparently inhibits GTPCH1 like BH4 by binding with GFRP; this inhibition is GFRP‐dependent. At higher concentrations, DAHP acts as a competitive inhibitor, binding at the GTPCH1 active site like GTP; this inhibition is GFRP‐independent (Xie et al., 1998, J Biol Chem 273: 21091). Using a reduction‐of‐function mutant in the worm GTPCH1 gene [cat‐4(e3015) – S148N], we find that DAHP reduces BH4 production, as assessed by a significant reduction in serotonin immunoreactivity in whole worms. Treatment with Phe significantly increases serotonin levels in cat‐4(e3015) worms. We are currently assessing the effects of mutations in gfrp‐1 on serotonin levels (hence BH4 levels) in single mutants and cat‐4; gfrp‐1 double mutants, and whether the effects of DAHP and Phe on BH4 synthesis are GFRP‐dependent.Support or Funding InformationSupported by a Univ of San Diego Faculty Research Grant and an endowment from the Fletcher Jones Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elevated production of the aromatic fragrance molecule, 2-phenylethanol, using Metschnikowia pulcherrima through both de novo and ex novo conversion in batch and continuous modes.

BACKGROUND2‐phenylethanol (2PE) is a fragrance molecule predominantly used in perfumes and the food industry. It can be made from petrochemicals inexpensively, however, this is unsuitable for most food applications. Currently, the main method of production for the bio‐derived compound is to extract the trace amounts found in rose petals, which is extremely costly. Potentially fermentation could provide an inexpensive, naturally sourced, alternative.RESULTSIn this investigation, 2PE was produced from the yeast Metschnikowia pulcherrima, optimised in flasks before scaling to 2 L batch and continuous operation. 2PE can be produced in high titres under de novo process conditions with up to 1500 mg L−1 achieved in a 2 L stirred bioreactor. This is the highest reported de novo titre to date, and achieved through high sugar loadings coupled with low nitrogen conditions. The process successfully ran in continuous mode also, with a concentration of 650 mg L−1 of 2PE being maintained. The 2PE production was further increased by the ex novo conversion of phenylalanine and semi‐continuous solid phase extraction from the supernatant. Under optimal conditions 14 000 mg L−1 of 2PE was produced.CONCLUSIONSThe work presented here offers a novel route to naturally sourced 2PE through a scalable fermentation with a robust yeast highly suited to industrial biotechnology. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Binding Pathways of Phenylalanine to the Dimeric Regulatory Domain of Human PAH Reveal a LID Gating Mechanism

Phenylalanine hydroxylase (PAH) is an enzyme that catalyzes the conversion of phenylalanine to tyrosine, and functions in humans to control free phenylalanine (Phe), an essential amino acid that is neurotoxic at elevated levels. Mutations in PAH can result in phenylketonuria, which is the most common inborn error of amino acid metabolism. The transition from resting-state PAH to activated PAH requires formation of a new intersubunit interface that can be stabilized by Phe binding in an allosteric manner; formation of the new protein:protein interface is coupled to exposure of the active site, thus activating the enzyme. A recent crystal structure of this interface bound to Phe enables new insight into phenylalanine binding mechanism from molecular dynamics (MD) simulations. We performed massively parallel explicit-solvent simulations on the [email protected] distributed computing platform to elucidate pathways and rates of binding of Phe to the interface, which is comprised of a dimer of ACT domains. Time-lagged independent component analysis (tICA) of binding trajectories suggest a conformational selection mechanism. Markov state models (MSMs) constructed from the trajectory data reveal a key loop motion which acts as a “gatekeeper” of allosteric ligand binding. Binding rates estimated by different methods (MSMs, Transition Path Theory and Bayesian inference) agree well with each other. These results warrant further MD studies of the conformational dynamics involved in regulation of PAH activity, the effect of disease-associated mutations, and suggest future directions in simulation-based drug discovery.

Biallelic Mutations in DNAJC12 Cause Hyperphenylalaninemia, Dystonia, and Intellectual Disability

Phenylketonuria (PKU, phenylalanine hydroxylase deficiency), an inborn error of metabolism, can be detected through newborn screening for hyperphenylalaninemia (HPA). Most individuals with HPA harbor mutations in the gene encoding phenylalanine hydroxylase (PAH), and a small proportion (2%) exhibit tetrahydrobiopterin (BH4) deficiency with additional neurotransmitter (dopamine and serotonin) deficiency. Here we report six individuals from four unrelated families with HPA who exhibited progressive neurodevelopmental delay, dystonia, and a unique profile of neurotransmitter deficiencies without mutations in PAH or BH4 metabolism disorder-related genes. In these six affected individuals, whole-exome sequencing (WES) identified biallelic mutations in DNAJC12, which encodes a heat shock co-chaperone family member that interacts with phenylalanine, tyrosine, and tryptophan hydroxylases catalyzing the BH4-activated conversion of phenylalanine into tyrosine, tyrosine into L-dopa (the precursor of dopamine), and tryptophan into 5-hydroxytryptophan (the precursor of serotonin), respectively. DNAJC12 was undetectable in fibroblasts from the individuals with null mutations. PAH enzyme activity was reduced in the presence of DNAJC12 mutations. Early treatment with BH4 and/or neurotransmitter precursors had dramatic beneficial effects and resulted in the prevention of neurodevelopmental delay in the one individual treated before symptom onset. Thus, DNAJC12 deficiency is a preventable and treatable cause of intellectual disability that should be considered in the early differential diagnosis when screening results are positive for HPA. Sequencing of DNAJC12 may resolve any uncertainty and should be considered in all children with unresolved HPA.

Fourteen years of newborn screening for phenylketonuria in Vojvodina

Introduction. Phenylketonuria is an inborn disorder of metabolism, a rare, hereditary disease caused by deficiency of phenylalanine hydroxylase enzyme necessary for conversion of phenylalanine into tyrosine. The aim of this study is to determine the incidence of hyperphenylalaninemia and classical phenylketonuria in population of the Autonomous Province of Vojvodina. Material and Methods. We performed retrospective analysis at Medical Genetics Service, the Institute for Youth and Health Care of Vojvodina and examined the clinical material of the previous fourteen years, during the interval from 2003-2016. The analysis of the obtained results was carried out using descriptive statistics. Results. During fourteen years, from 01.01.2003 to 31.12.2016, 27 newborns with hyperphenylalaninemia were detected, and the incidence of hyperphenylalaninemia in the Autonomous Province of Vojvodina was 1: 9.525. Classical phenylketonuria was detected in 15 persons during indicated period, and the incidence was 1:17.143. Conclusion. Phenylketonuria is a hereditary disease whose adverse effects can be avoided, if it is recognized in time, and if recommended treatment measures are adequately applied, thereby improving the quality of life of persons affected by the disease as well as the whole family, that is facilitated by the introduction and implementation of neonatal screening.

Pathway Analysis and Metabolites Identification by Metabolomics of Etiolation Substrate from Fresh-Cut Chinese Water Chestnut (Eleocharis tuberosa).

Fresh-cut Chinese water chestnuts (CWC) turn yellow after being peeled, reducing their shelf life and commercial value. Metabolomics, the systematic study of the full complement of small molecular metabolites, was useful for clarifying the mechanism of fresh-cut CWC etiolation and developing methods to inhibit yellowing. In this study, metabolic alterations associated with etiolation at different growth stages (0 day, 2 days, 3 days, 4 days, 5 days) from fresh-cut CWC were investigated using LC–MS and analyzed by pattern recognition methods (principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and orthogonal projection to latent structures-discriminant analysis (OPLS-DA)). The metabolic pathways of the etiolation molecules were elucidated. The main metabolic pathway appears to be the conversion of phenylalanine to p-coumaroyl-CoA, followed by conversion to naringenin chalcone, to naringenin, and naringenin then following different pathways. Firstly, it can transform into apigenin and its derivatives; secondly, it can produce eriodictyol and its derivatives; and thirdly it can produce dihydrokaempferol, quercetin, and myricetin. The eriodictyol can be further transformed to luteolin, cyanidin, dihydroquercetin, dihydrotricetin, and others. This is the first reported use of metabolomics to study the metabolic pathways of the etiolation of fresh-cut CWC.

Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering.

Mammalian phenylalanine hydroxylase (PheH) is an allosteric enzyme that catalyzes the first step in the catabolism of the amino acid phenylalanine. Following allosteric activation by high phenylalanine levels, the enzyme catalyzes the pterin-dependent conversion of phenylalanine to tyrosine. Inability to control elevated phenylalanine levels in the blood leads to increased risk of mental disabilities commonly associated with the inherited metabolic disorder, phenylketonuria. Although extensively studied, structural changes associated with allosteric activation in mammalian PheH have been elusive. Here, we examine the complex allosteric mechanisms of rat PheH using X-ray crystallography, isothermal titration calorimetry (ITC), and small-angle X-ray scattering (SAXS). We describe crystal structures of the preactivated state of the PheH tetramer depicting the regulatory domains docked against the catalytic domains and preventing substrate binding. Using SAXS, we further describe the domain movements involved in allosteric activation of PheH in solution and present the first demonstration of chromatography-coupled SAXS with Evolving Factor Analysis (EFA), a powerful method for separating scattering components in a model-independent way. Together, these results support a model for allostery in PheH in which phenylalanine stabilizes the dimerization of the regulatory domains and exposes the active site for substrate binding and other structural changes needed for activity.

Effect of experimental phenylketonuria on the development of skin of prenatel and newborn fetuses.

Phenylketonuria (PKU) is a genetic disorder that is characterized by an inability of the body to utilize the essential amino acid, phenylalanine. The disease results from a deficiency in phenylalanine hydroxylase, the enzyme catalyzing the conversion of phenylalanine to tyrosine. The present work studied the Effect of experimental phenylketonuria on the development of skin of prenatel and newborn fetuses.The disease was induced in pregnant rats by daily intragastric administration of 30 mg. DL-a-methylphenylalanine/kg body weight plus 60 mg/kg body weight at 12 h intervals throughout pregnancy till parturition. Treatment with alpha-methylphenylalanine/phenylalanine resulted in significant decrease of accumulated body weight gain during pregnancy as well as exhibited marked growth retardation of prenatal fetuses and delivered newborn. The growth retarded fetuses was manifested by decreased body weight, malformed both fore -& hind limb, oedematous skin & superficial hematomas widely spreads in different parts of the body. Vibrissae skin hair were characterized by curved free ends as well as reduction of their size and length. Cornification was retarded . The epidermis attained a considerable thinning with delayed differentiation of epidermal cell layers.The growing hair follicles lacked normal characteristic appearance. These results suggested that exposure of the fetus to high plasma concentrations of phenylalanine caused a delay in the biochemical maturation of the fetal rat.

Biochemical Evaluation of Phenylalanine Ammonia Lyase from Endemic Plant Cyathobasis fruticulosa (Bunge) Aellen. for the Dietary Treatment of Phenylketonuria.

Enzyme substitution therapy with the phenylalanine ammonia lyase (PAL) is a new approach to the treatment of patients with phenylketonuria (PKU). This enzyme is responsible for the conversion of phenylalanine to trans-cinnamic acid. We assessed the PAL enzyme of the endemic plant Cyathobasis fruticulosa (Bunge) Aellen. for its possible role in the dietary treatment of PKU. The enzyme was found to have a high activity of (64.9±0.1) U/mg, with the optimum pH, temperature and buffer (Tris-HCl and l-phenylalanine) concentration levels of pH=8.8, 37 °C and 100 mM, respectively. Optimum enzyme activity was achieved at pH=4.0 and 7.5, corresponding to pH levels of gastric and intestinal juice, and NaCl concentration of 200 mM. The purification of the enzyme by 1.87-fold yielded an activity of 98.6 U/mg. PAL activities determined by HPLC analyses before and after purification were similar. Two protein bands, one at 70 and the other at 23 kDa, were determined by Western blot analysis of the enzyme. This enzyme is a potential candidate for serial production of dietary food and biotechnological products.

Phenylketonuria (PKU): A problem solved?

Phenylketonuria (PKU) is a rare metabolic disorder characterized by impaired conversion of phenylalanine (Phe) to tyrosine. If left untreated, the resultant accumulation of excess blood Phe can cause physiological, neurological, and intellectual disabilities. The National PKU Alliance (NPKUA) conducted a survey of its membership to assess current health status and interest in new treatments for PKU. Of the 625 survey respondents, less than half (46.7%) reported blood Phe within (120–360μmol/L) — the range recommended by the American College of Medical Genetics and Genomics (ACMG). The survey results also showed that younger (≤18years) individuals were about 3-times as successful in keeping their blood Phe concentrations within the recommended clinical range compared with adults. Blood Phe over 360μmol/L was reported in one-quarter (25.5%) of ≤18year old individuals and almost two-thirds (61.5%) of adults. A little more than half (51.7%) of respondents reported having difficulty in managing their PKU, including the maintenance of a Phe-restricted diet. Individuals with PKU desire new treatments that would allow them to increase their intake of natural protein, discontinue or reduce their intake of medical foods (medical formula and foods modified to be low in protein), improve their mental health (including a reduction in depression and anxiety), and a reduction of their blood Phe concentrations. Respondents preferred oral administration of any newly developed therapies and, in general, disliked therapeutic injections. Injections at home were preferred over injections at a clinic. Payers, government agencies, clinicians, and industry partners should consider patient input when developing and approving new therapies and treatments for PKU.

Application of Pseudomonas fluorescens to Blackberry under Field Conditions Improves Fruit Quality by Modifying Flavonoid Metabolism.

Application of a plant growth promoting rhizobacterium (PGPR), Pseudomonas fluorescens N21.4, to roots of blackberries (Rubus sp.) is part of an optimised cultivation practice to improve yields and quality of fruit throughout the year in this important fruit crop. Blackberries are especially rich in flavonoids and therefore offer potential benefits for human health in prevention or amelioration of chronic diseases. However, the phenylpropanoid pathway and its regulation during ripening have not been studied in detail, in this species. PGPR may trigger flavonoid biosynthesis as part of an induced systemic response (ISR) given the important role of this pathway in plant defence, to cause increased levels of flavonoids in the fruit. We have identified structural genes encoding enzymes of the phenylpropanoid and flavonoid biosynthetic pathways catalysing the conversion of phenylalanine to the final products including flavonols, anthocyanins and catechins from blackberry, and regulatory genes likely involved in controlling the activity of pathway branches. We have also measured the major flavonols, anthocyanins and catechins at three stages during ripening. Our results demonstrate the coordinated expression of flavonoid biosynthetic genes with the accumulation of anthocyanins, catechins, and flavonols in developing fruits of blackberry. Elicitation of blackberry plants by treatment of roots with P.fluorescens N21.4, caused increased expression of some flavonoid biosynthetic genes and an accompanying increase in the concentration of selected flavonoids in fruits. Our data demonstrate the physiological mechanisms involved in the improvement of fruit quality by PGPR under field conditions, and highlight some of the genetic targets of elicitation by beneficial bacteria.

Role of GTP-CHI links PAH and TH in melanin synthesis in silkworm, Bombyx mori

In insects, pigment patterns are formed by melanin, ommochromes, and pteridines. Here, the effects of pteridine synthesis on melanin formation were studied using 4th instar larvae of a wild-type silkworm strain, dazao (Bombyx mori), with normal color and markings. Results from injected larvae and in vitro integument culture indicated that decreased activity of guanosine triphosphate cyclohydrolase I (GTP-CH I, a rate-limiting enzyme for pteridine synthesis), lowers BH4 (6R-l-erythro-5,6,7,8-tetrahydrobiopterin, a production correlated with GTP-CH I activity) levels and eliminates markings and coloration. The conversion of phenylalanine and tyrosine to melanin was prevented when GTP-CH I was inhibited. When BH4 was added, phenylalanine was converted to tyrosine, and the tyrosine concentration increased. Tyrosine was then converted to melanin to create normal markings and coloration. Decreasing GTP-CH I activity did not affect L-DOPA (3,4-l-dihydroxyphenylalanine). GTP-CH I affected melanin synthesis by generating the BH4 used in two key reaction steps: (1) conversion of phenylalanine to tyrosine by PAH (phenylalanine hydroxylase) and (2) conversion of tyrosine to L-DOPA by TH (tyrosine hydroxylase). Expression profiles of BmGTPCH Ia, BmGTPCH Ib, BmTH, and BmPAH in the integument were consistent with the current findings.

Minimally invasive (13)C-breath test to examine phenylalanine metabolism in children with phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by deficiency of hepatic phenylalanine hydroxylase (PAH) leading to increased levels of phenylalanine in the plasma. Phenylalanine levels and phenylalanine hydroxylase (PAH) activity monitoring are currently limited to conventional blood dot testing. 1-(13)C-phenylalanine, a stable isotope can be used to examine phenylalanine metabolism, as the conversion of phenylalanine to tyrosine occurs in vivo via PAH and subsequently releases the carboxyl labeled (13)C as (13)CO2 in breath. Our objective was to examine phenylalanine metabolism in children with PKU using a minimally-invasive 1-(13)C-phenylalanine breath test ((13)C-PBT). Nine children (7 M: 2 F, mean age 12.5 ± 2.87 y) with PKU participated in the study twice: once before and once after sapropterin supplementation. Children were provided 6 mg/kg oral dose of 1-(13)C-phenylalanine and breath samples were collected at 20 min intervals for a period of 2h. Rate of CO2 production was measured at 60 min post-oral dose using indirect calorimetry. The percentage of 1-(13)C-phenylalanine exhaled as (13)CO2 was measured over a 2h period. Prior to studying children with PKU, we tested the study protocol in healthy children (n = 6; 4M: 2F, mean age 10.2 ± 2.48 y) as proof of principle. Production of a peak enrichment (Cmax) of (13)CO2 (% of dose) in all healthy children occurred at 20 min ranging from 17-29% of dose, with a subsequent return to ~5% by the end of 2h. Production of (13)CO2 from 1-(13)C-phenylalanine in all children with PKU prior to sapropterin treatment remained low. Following sapropterin supplementation for a week, production of (13)CO2 significantly increased in five children with a subsequent decline in blood phenylalanine levels, suggesting improved PAH activity. Sapropterin treatment was not effective in three children whose (13)CO2 production remained unchanged, and did not show a reduction in blood phenylalanine levels and improvement in dietary phenylalanine tolerance. Our study shows that the (13)C-PBT can be a minimally invasive, safe and reliable measure to examine phenylalanine metabolism in children with phenylketonuria. The breath data are corroborated by blood phenylalanine levels in children who had increased responses in (13)CO2 production, as reviewed post-hoc from clinical charts.

Production of tyrosine through phenylalanine hydroxylation bypasses the intrinsic feedback inhibition in Escherichia coli.

Tyrosine is a proteinogenic aromatic amino acid that is often used as a supplementof food and animal feed, as well as a (bio-)synthetic precursor to various pharmaceutically or industrially important molecules. Extensive metabolic engineering efforts have been made towards the efficient and cost-effective microbial production of tyrosine. Conventional strategies usually focus on eliminating intrinsic feedback inhibition and redirecting carbon flux into the shikimate pathway. In this study, we found that continuous conversion of phenylalanine into tyrosine by the action of tetrahydromonapterin (MH4)-utilizing phenylalanine 4-hydroxylase (P4H) can bypass the feedback inhibition in Escherichia coli, leading to tyrosine accumulation in the cultures. First, expression of the P4H from Xanthomonas campestris in combination with an MH4 recycling system in wild-type E. coli allowed the strain to accumulate tyrosine at 262mg/L. On this basis, enhanced expression of the key enzymes associated with the shikimate pathway and the MH4 biosynthetic pathway resulted in the elevation of tyrosine production up to 401mg/L in shake flasks. This work demonstrated a novel approach to tyrosine production and verified the possibility to alleviate feedback inhibition by creating a phenylalanine sink.