Hydroxylation Of Phenylalanine Research Articles

Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates - A Substudy.

BackgroundBased on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates.ObjectiveTo assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake.MethodsA randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d) or low protein (0.4 g protein/kg/d) energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat) for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y) were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans.ResultsAfter 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;P<0.001). Whole-body protein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;P<0.03), synthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;P<0.01) and phenylalanine hydroxylation rates (4.1±0.6 vs 2.7±0.6 μmol phenylalanine/kg/h;P<0.001) were significantly higher in the high vs low protein group. Basal muscle protein synthesis rates were maintained on a low vs high protein diet (0.042±0.01 vs 0.045±0.01%/h;P = 0.620).ConclusionsIn the overnight fasted state, adaptation to a low-protein intake (0.4 g/kg/d) does not result in a more negative whole-body protein balance and does not lower basal muscle protein synthesis rates when compared to a high-protein intake.Trial RegistrationClinicaltrials.gov NCT01551238.

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.

Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein’s regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The kcat/Kphe value is down 104 for the mutant enzyme, and the Km value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

Disturbed biopterin and folate metabolism in the Qdpr-deficient mouse

Quinonoid dihydropteridine reductase (QDPR) catalyzes the regeneration of tetrahydrobiopterin (BH4), a cofactor for monoamine synthesis, phenylalanine hydroxylation and nitric oxide production. Here, we produced and analyzed a transgenic Qdpr−/− mouse model. Unexpectedly, the BH4 contents in the Qdpr−/− mice were not decreased and even increased in some tissues, whereas those of the oxidized form dihydrobiopterin (BH2) were significantly increased. We demonstrated that unlike the wild-type mice, dihydrofolate reductase regenerated BH4 from BH2 in the mutants. Furthermore, we revealed wide alterations in folate-associated metabolism in the Qdpr−/− mice, which suggests an interconnection between folate and biopterin metabolism in the transgenic mouse model.

An isotope dilution model for partitioning phenylalanine and tyrosine uptake by the mammary gland of lactating dairy cows

An isotope dilution model for partitioning phenylalanine and tyrosine uptake by the mammary gland of the lactating dairy cow is constructed and solved in the steady state. The model contains four intracellular and four extracellular pools and conservation of mass principles are applied to generate the fundamental equations describing the behaviour of the system. The experimental measurements required for model solution are milk secretion and plasma flow rate across the gland in combination with phenylalanine and tyrosine concentrations and plateau isotopic enrichments in arterial and venous plasma and free and protein bound milk during a constant infusion of [1-13C]phenylalanine and [2,3,5,6-2H]tyrosine tracer. If assumptions are made, model solution enables determination of steady state flows for phenylalanine and tyrosine inflow to the gland, outflow from it and bypass, and flows representing the synthesis and degradation of constitutive protein and phenylalanine hydroxylation. The model is effective in providing information about the fates of phenylalanine and tyrosine in the mammary gland and could be used as part of a more complex system describing amino acid metabolism in the whole ruminant.

Systemic arginine depletion: effects on protein synthesis and blood pressure in mice (258.4)

Arginine (Arg) is a semi‐essential amino acid involved in protein synthesis (PS) and blood pressure (BP) regulation. Arg deiminase (ADI) is a bacterial enzyme that hydrolyses Arg imino group to yield citrulline (Cit) and NH3. Pegylated ADI (ADI‐PEG) administration in animals and humans results in the depletion of circulating Arg. To investigate the effect of ADI‐PEG on PS and BP, ADI‐PEG or Saline (Sal) were administered i.m. to male mice. Plasma Arg concentration was negligible (&lt;1; Sal 80 µmol/L), but Cit increased several fold (1100; Sal 80 µmol/L) in ADI‐PEG treated mice. Arg and Cit fluxes were greater in ADI‐PEG mice (Arg 543 vs 910, Cit 126 vs 402 µmol•kg‐1•h‐1, Sal and ADI‐PEG, resp.). The conversion of Arg to Cit, usually used as a proxy for NO production, increased from 4.1 to 496 µmol•kg‐1•h‐1, demonstrating the activity of the deiminase. Phenylalanine and tyrosine fluxes and phenylalanine hydroxylation were not different between the two treatments, indicating no changes in whole body protein kinetics. Protein fractional synthesis rate was not different for most tissues, but it was lower in the brain, thymus and testicles of ADI‐PEG treated mice. 14C Cit incorporation showed an identical pattern. Mean BP was reduced in ADI‐PEG treated mice (105 vs Sal 118 mm Hg, P&lt; 0.008), despite the virtual absence of Arg in blood. These results demonstrate the capacity of many cell types to derive ARG from Cit to meet their needs.Grant Funding Source: Supported by USDA/ARS Cooperative Agreement No 58‐6250‐6‐001

Adaptive regulation of amino acid metabolism on early parenteral lipid and high-dose amino acid administration in VLBW infants – A randomized, controlled trial

An anabolic state can be achieved upon intravenous amino acid administration during the immediate postnatal phase despite a low energy intake. The optimal dosing of amino acid and energy intake has yet to be established. The aim was to quantify the efficacy of early initiation of parenteral lipids and increased amounts of amino acids on metabolism and protein accretion in very low birth weight infants. 28 very low birth weight infants were randomized to receive parenteral nutrition with glucose and either 2.4gamino acids/(kg·d) (control group), 2.4gamino acids/(kg·d) plus 2-3glipid/(kg·d) (AA+lipid group), or 3.6gamino acids/(kg·d) plus 2-3glipid/(kg·d) (high AA+lipid group) from birth onward. On postnatal day 2, we performed a stable isotope study with [1-(13)C]phenylalanine, [ring-D4]tyrosine, [U-(13)C6,(15)N]leucine, and [methyl-D3]α-ketoisocaproic acid to quantify intermediate amino acid metabolism. The addition of lipids only had no effect on phenylalanine metabolism, whereas the addition of both lipids and additional amino acids increased the amount of phenylalanine used for protein synthesis. In addition, high amino acid intake significantly increased the rate of hydroxylation of phenylalanine to tyrosine, increasing the availability of tyrosine for protein synthesis. However, it also increased urea concentrations. Increasing energy intake from 40 to 60kcal/(kg·d) did not increase protein efficiency as measured by phenylalanine kinetics. The leucine data were difficult to interpret due to the wide range of results and inconsistency in the data between the phenylalanine and leucine models. High amino acid and energy intakes from birth onwards result in a more anabolic state in very low birth weight infants, but at the expense of higher urea concentrations, which reflects a higher amino acid oxidation. Long-term outcome data should reveal whether this policy deserves routine implementation. This trial was registered at www.trialregister.nl, trial number NTR1445, name Nutritional Intervention for Preterm Infants-2.

UNNOTICED IN CHILDHOOD, DISABLING IN ADULTHOOD: A PROGRESSIVE BUT TREATABLE DISEASE…

A 64 year old Caucasian man presented with subtle cognitive decline of uncertain duration and a six month history of progressive difficulty mobilising with falls. He reported increasing stiffness in...

Regulation of phenylalanine hydroxylase: Conformational changes upon phosphorylation detected by H/D exchange and mass spectrometry

The enzyme phenylalanine hydroxylase catalyzes the hydroxylation of excess phenylalanine in the liver to tyrosine. The enzyme is regulated allosterically by phenylalanine and by phosphorylation of Ser16. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into any structural change upon phosphorylation. Peptides in both the catalytic and regulatory domains show increased deuterium incorporation into the phosphorylated protein. Deuterium is incorporated into fewer peptides than when the enzyme is activated by phenylalanine, and the incorporation is slower. This establishes that the conformational change upon phosphorylation of phenylalanine hydroxylase is different from and less extensive than that upon phenylalanine activation.

Mutagenesis of a Specificity-Determining Residue in Tyrosine Hydroxylase Establishes That the Enzyme Is a Robust Phenylalanine Hydroxylase but a Fragile Tyrosine Hydroxylase

The aromatic amino acid hydroxylases tyrosine hydroxylase (TyrH) and phenylalanine hydroxylase (PheH) have essentially identical active sites; however, PheH is nearly incapable of hydroxylating tyrosine, while TyrH can readily hydroxylate both tyrosine and phenylalanine. Previous studies have indicated that Asp425 of TyrH is important in determining the substrate specificity of that enzyme [Daubner, S. C., Melendez, J., and Fitzpatrick, P. F. (2000) Biochemistry 39, 9652-9661]. Alanine-scanning mutagenesis of amino acids 423-427, a mobile loop containing Asp425, shows that only mutagenesis of Asp425 alters the activity of the enzyme significantly. Saturation mutagenesis of Asp425 results in large (up to 10(4)) decreases in the V(max) and V(max)/K(tyr) values for tyrosine hydroxylation, but only small decreases or even increases in the V(max) and V(max)/K(phe) values for phenylalanine hydroxylation. The decrease in the tyrosine hydroxylation activity of the mutant proteins is due to an uncoupling of tetrahydropterin oxidation from amino acid hydroxylation with tyrosine as the amino acid substrate. In contrast, with the exception of the D425W mutant, the extent of coupling of tetrahydropterin oxidation and amino acid hydroxylation is unaffected or increases with phenylalanine as the amino acid substrate. The decrease in the V(max) value with tyrosine as the substrate shows a negative correlation with the hydrophobicity of the amino acid residue at position 425. The results are consistent with a critical role of Asp425 being to prevent a hydrophobic interaction that results in a restricted active site in which hydroxylation of tyrosine does not occur.

Pitfalls in phenylalanine loading test in the diagnosis of dopa-responsive dystonia

Phenylalanine (Phe) loading test is a useful tool in the differential diagnosis of dopa-responsive dystonia due to autosomal dominant or recessive GTP cyclohydrolase I (GTPCH) deficiency or autosomal recessive sepiapterin reductase (SR) deficiency. In these patients hepatic phenylalanine hydroxylase system is compromised due to subnormal tetrahydrobiopterin (BH4) levels and hydroxylation of phenylalanine (Phe) to tyrosine (Tyr) is reduced with elevated Phe/Tyr ratio 1–2h after oral Phe administration (100mg/kg bw) administration. In healthy persons there is only a modest increase in Tyr production and blood Phe normalizes after 4h. We report on a challenge with Phe (100mg/kg bw) in a patient with dopa-responsive dystonia while on therapy with BH4 and l-dopa. During Phe challenge Phe concentration remained below the normal range while a transient mild hypertyrosinemia was observed, leading to an extremely low Phe/Tyr ratio. A repeated test, after BH4 withdrawal, reversed the findings and resulted normal. These data suggest activation of hepatic phenylalanine hydroxylase by BH4. Thus, the Phe loading test should not be performed during substitution with BH4.

Importance of early diagnosis of phenylketonuria in women and control of phenylalanine levels during pregnancy

The phenylalanine hydroxylase (PAH) in the liver hydroxylates phenylalanine from the diet. Fetuses depend for the hydroxylation of phenylalanine the maternal metabolism , fetal maturity does not come until week 26. Though the women with PAH deficiency (phenylketonuria, PKU) not adequately hydroxylate phenylalanine diet so their blood levels are high. Fenilalaninemia levels are considered neurotoxic teratogenic and above 360 umol/L (N < 120). Pregnant women should strictly follow PKU dietary treatment and/or drug to maintain levels of fenilalaninemia < 180 umol/L and avoid the teratogenic complications in the fetus (Hyperphenylalaninaemias Maternal Fetal Syndrome), as the case presented. We recommend discarding Phenylketonuria in women who have not been done a neonatal screening and/or have abortions, children with microcephaly, cardiac or renal malformations.

Meta-Tyrosine in Festuca rubra ssp. commutata (Chewings fescue) is synthesized by hydroxylation of phenylalanine

m-Tyrosine is a non-protein amino acid that is structurally similar to the common protein amino acids p-tyrosine and phenylalanine. Copious amounts of m-tyrosine can be found in root exudates of the fine fescue cultivar, Festuca rubra L. ssp. commutata (Chewings fescue). The phytotoxicity of m-tyrosine may contribute to the allelopathic potential of F. rubra. m-Tyrosine in Euphorbia myrsinites (donkey-tail spurge), was previously shown to be synthesized via transamination of m-hydroxyphenylpyruvate. Here we show that m-tyrosine biosynthesis in F. rubra occurs through direct hydroxylation of phenylalanine in the root tips, perhaps through the activity of a cytochrome P450 enzyme. Hence, E. myrsinites and F. rubra, the only two plant species known to produce m-tyrosine, use distinct biosynthetic pathways that likely arose independently in evolutionary history.

Nitration of Tyrosine 10 Critically Enhances Amyloid β Aggregation and Plaque Formation

Part of the inflammatory response in Alzheimer's disease (AD) is the upregulation of the inducible nitric oxide synthase (NOS2) resulting in increased NO production. NO contributes to cell signaling by inducing posttranslational protein modifications. Under pathological conditions there is a shift from the signal transducing actions to the formation of protein tyrosine nitration by secondary products like peroxynitrite and nitrogen dioxide. We identified amyloid β (Aβ) as an NO target, which is nitrated at tyrosine 10 (3NTyr(10)-Aβ). Nitration of Aβ accelerated its aggregation and was detected in the core of Aβ plaques of APP/PS1 mice and AD brains. NOS2 deficiency or oral treatment with the NOS2 inhibitor L-NIL strongly decreased 3NTyr(10)-Aβ, overall Aβ deposition and cognitive dysfunction in APP/PS1 mice. Further, injection of 3NTyr(10)-Aβ into the brain of young APP/PS1 mice induced β-amyloidosis. This suggests a disease modifying role for NOS2 in AD and therefore represents a potential therapeutic target.

Folate Status and Depressive Symptoms in Reproductive-Age Women

Folate is a water-soluble B-vitamin necessary for the proper biosynthesis of the monoamine neurotransmitters serotonin, epinephrine, and dopamine. Epidemiological and biological evidence suggests that low folate appears to be most closely linked to depressive disorders among individuals with epilepsy, people suffering from neurological and psychiatric problems, as well as the elderly. Folic acid influences the rate of synthesis of the neurotransmitters dopamine, norepinephrine, and serotonin and acts as a cofactor in the hydroxylation of phenylalanine and tryptophan. A disturbance of biogenic amine metabolism may lead to various psychiatric disorders. Folic acid deficiency may contribute to pathogenesis of neuropsychiatric disorders such as mental confusion, memory changes, cognitive slowing, and mood disorder. However, since almost all published studies have been conducted with the elderly or with persons with dementia disease, the effects of folate on the mental health of people of reproductive age are not yet known. Findings from this review suggest that low folate status can be a key factor in the expression of depressive symptoms. However, it is difficult to evaluate whether folate deficiency is a substantial contributor to depressive symptoms in reproductive-age women in scan data compared to the numerous data for older women. A critical goal is for women to make behavior changes for good nutritional status before, during, and beyond pregnancy, as this may lead to improved mental and physical health in later life for both themselves and their family members.

Rate of Phenylalanine Hydroxylation in Healthy School-Aged Children

Hydroxylation of phenylalanine to tyrosine is the first and rate-limiting step in phenylalanine catabolism. Currently, there are data on the rate of phenylalanine hydroxylation in infants and adults but not in healthy children. Thus, the aim of the study reported here was to measure the rate of phenylalanine hydroxylation and oxidation in healthy school-aged children both when receiving diets with and without tyrosine. In addition, hydroxylation rates calculated from the isotopic enrichments of amino acids in plasma and in very LDL apoB-100 were compared. Eight healthy 6- to 10-y-old children were studied while receiving a control and again while receiving a tyrosine-free diet. Phenylalanine flux, hydroxylation, and oxidation were determined by a standard tracer protocol using oral administration of ¹³C-phenylalanine and ²H₂-tyrosine for 6 h. Phenylalanine hydroxylation rate of children fed a diet devoid of tyrosine was greater than that of children fed a diet containing tyrosine (40.25 ± 5.48 versus 29.55 ± 5.35 μmol · kg⁻¹ · h⁻¹; p < 0.01). Phenylalanine oxidation was not different from phenylalanine hydroxylation regardless of dietary tyrosine intake, suggesting that phenylalanine converted to tyrosine was mainly oxidized. In conclusion, healthy children are capable of converting phenylalanine to tyrosine, but the need for tyrosine cannot be met by providing extra phenylalanine.

Evidence for a high-spin Fe(IV) species in the catalytic cycle of a bacterial phenylalanine hydroxylase.

Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mössbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme-(57)Fe(II)-phenylalanine-6-methyltetrahydropterin complex with O(2) reveal the accumulation of an intermediate at short reaction times (20-100 ms). The Mössbauer parameters of the intermediate (δ = 0.28 mm/s, and |ΔE(Q)| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme-substrate complex reacts with oxygen to form an Fe(IV)═O intermediate with a rate constant of 19 mM(-1) s(-1), the Fe(IV)═O intermediate hydroxylates phenylalanine with a rate constant of 42 s(-1), and rate-limiting product release occurs with a rate constant of 6 s(-1) at 5 °C.

Advances and challenges in phenylketonuria

Phenylketonuria (PKU; OMIM 262600), one of the most common inborn errors of metabolism, is caused by recessively inherited deficiency of the enzyme phenylalanine hydroxylase (PAH; EC 1.14.16.1) (Blau et al. 2010). PAH catalyses the irreversible hydroxylation of phenylalanine (Phe) to tyrosine. PAH is expressed primarily in the liver but also in the kidney and pancreas, and its activity requires the unconjugated pterin co-factor, tetrahydrobiopterin (BH4). PAH deficiency causes hyperphenylalaninaemia, but hyperphenylalaninaemia can also be caused by inherited deficiency of enzymes involved in BH4 synthesis or recycling (Blau et al. 2010). Chronic, untreated, severe hyperphenylalaninaemia in infants and children leads to seizures and mental retardation. Newborn screening and early initiation of PKU therapy has eliminated the major manifestations of the disease, but shortcomings in our current therapeutic approach remain. Contemporary therapy for PKU is centered upon tight restriction of dietary Phe intake and requires supplementation with special medical foods that supply sufficient essential amino acids and energy from fat and carbohydrate. Institution and maintenance of the PKU diet are difficult, and the required medical foods are often unpalatable. Dietary therapy is recommended for life (Anon 2001), but non-compliance with the dietary prescription is commonplace, particularly during adolescence and adulthood. Hyperphenylalaninaemia in adults is often associated with attention problems, mood instability and poor job performance. Chronically elevated Phe may cause a progressive neurodegenerative disorder affecting white matter that leads to seizures and gait disturbance. Finally, untreated maternal hyperphenylalaninaemia during pregnancy is the only teratogen guaranteed to cause birth defects, which include microcephaly, mental retardation and congenital heart disease (Levy et al. 2003), the so-called maternal PKU syndrome. For all these reasons, novel alternative therapies for PKU are being sought. Phe itself, accumulating to extremely high levels, is primarily responsible for the phenotype associated with untreated PKU. Accumulation of Phe in blood (normally less than 150 μM but often over 2,000 μM in untreated patients) severely interferes with growth and maturation of the developing brain, although the exact molecular mechanism by which this occurs has yet to be elucidated. Over 50 years of experience with PKU treatment based upon the restriction of dietary Phe intake have firmly demonstrated the link between clinical outcome and blood Phe levels (Azen et al. 1996). So, any treatment approach that results in decreased blood Phe levels will ameliorate the PKU phenotype. BH4 supplementation is a novel therapeutic approach that is effective in a subset of individuals with PKU (Blau 2010; Harding 2010). In addition to PAH, BH4 is a cofactor Presented in part at the 1st European Phenylketonuria Group Symposium, January, 2009 in Barcelona, Spain (supported by Serono Symposia International Foundation).

Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase

The hydroxylation of phenylalanine to tyrosine by the liver enzyme phenylalanine hydroxylase is regulated by the level of phenylalanine. Whether there is a distinct allosteric binding site for phenylalanine outside of the active site has been unclear. The enzyme contains an N-terminal regulatory domain that extends through Thr117. The regulatory domain of rat phenylalanine hydroxylase was expressed in Escherichia coli. The purified protein behaves as a dimer on a gel filtration column. In the presence of phenylalanine, the protein elutes earlier from the column, consistent with a conformational change in the presence of the amino acid. No change in elution is seen in the presence of the non-activating amino acid proline. 1H–15N HSQC NMR spectra were obtained of the 15N-labeled protein alone and in the presence of phenylalanine or proline. A subset of the peaks in the spectrum exhibits chemical shift perturbation in the presence of phenylalanine, consistent with binding of phenylalanine at a specific site. No change in the NMR spectrum is seen in the presence of proline. These results establish that the regulatory domain of phenylalanine hydroxylase can bind phenylalanine, consistent with the presence of an allosteric site for the amino acid.

Bioconversion of AS1387392: bioconversion studies involving Amycolatopsis azurea JCM 3275

We screened actinomycetes capable of converting AS1387392 to AS1429716 and identified those strains capable of hydroxylation. Amycolatopsis azurea JCM 3275 was found to be a particularly efficient strain, capable of converting AS1387392 to AS1429716, with a yield of 44% after 9 h. This strain can metabolize not only the hydroxylation of phenylalanine at the meta and para positions but also the reduction of hydroxyketones, as shown by the isolation of bioconversion products. Examination of more suitable conversion conditions showed that pH 7.8 and 25 °C were the optimum pH and temperature for bioconversion, respectively. We also demonstrated the effect of carbon and nitrogen sources in the culture media on hydroxylation. Using this strain, we were able to efficiently produce AS1429716 as a chemical template. Further derivatization studies may provide more effective, safer immunosuppressants than those that are currently on-market.