Dependent Phenylalanine Research Articles

In silico thermodynamics stability change analysis involved in BH4 responsive mutations in phenylalanine hydroxylase: QM/MM and MD simulations analysis

The mammalian tetrahydrobiopterin (BH4)-dependent phenylalanine hydroxylases (PAH), involved in important metabolic pathways of phenylalanine, belong to non-heme iron-containing aromatic acid hydroxylases’ enzyme (AAH) family. AAHs utilize BH4 as protein co-factor and thus promote hydroxylation reactions of their substrates. Any alterations in BH4 -mediated AAH’s pathway or mutations in these enzymes are responsible for various disorders, and thus highlights the importance of mutational analysis to assess the effect on their biosynthetic pathways. Our present studies are aimed at single-site mutations in PAH that lead to thermodynamic stability change upon folding and further validation of designed non-reduced BH2 designed co-factors. We have presented single-site mutational analysis of PAH where single-site mutations have been identified from known literature. Further, in silico studies with the PAH, in silico mutant PAH, and crystallized known mutant A313T forms, involved QM/MM and Molecular Dynamics (MD) simulations analysis. The modified co-factor A showed high affinity with PAH and all mutant PAH with high G-score of −14.851. The best pose high affinity co-factor A subjected to QM/MM optimization which leads to square-pyramidal coordination of non-heme active site. The structural and energetic information obtained from the production phase of 20 ns MD simulation of co-factor-metalloprotein complex results helped to understand the binding mode and involvement of three molecules throughout the reaction pathways’ catalysis of PAH. The free energies of binding (dG) of A were found to be −68.181 kcal/mol and −72.249 for 1DMW and 1TDW for A313T mutant. Binding of Co-factor A do not perturb the coordination environment of iron at the active site which resides in 2-Histdine and 1-Glutamate triad, and may enhance the percentage response towards co-factor-mediated therapy.

Regulation of melanogenesis – controversies and new concepts

Despite many efforts, regulation of skin and hair pigmentation is still not fully understood. This article focuses mainly on controversial aspects in pigment cell biology which have emerged over the last decade. The central role of tyrosinase as the key enzyme in initiation of melanogenesis has been closely associated with the 6BH4 dependent phenylalanine hydroxylase (PAH) and tyrosine hydroxylase isoform I (THI) providing evidence for an old concept of the three enzyme theory in the initiation of the pigmentation process. In this context, it is noteworthy that intracellular L-phenylalanine uptake and turnover to L-tyrosine via PAH is vital for substrate supply of THI and tyrosinase. While PAH acts in the cytosol of melanocytes, THI and tyrosinase are sitting side by side in the melanosomal membrane. THI at low pH provides L-3,4-hydroxyphenylalanine L-DOPA which in turn is required for activation of met-tyrosinase. After an intramelanosomal pH change, possibly by the p-protein, has taken place, tyrosinase is subject to control by 6/7BH4 and the proopiomelanocortin (POMC) peptides alpha-MSH melanocyte stimulating hormone and beta-MSH in a receptor independent manner. cAMP is required for the activation of microphthalmia-associated transcription factor to induce expression of tyrosinase, for transcription of THI and for activation of PAH. The redundancy of the cAMP signal is discussed. Finally, we propose a novel mechanism involving H2O2 in the regulation of tyrosinase via p53 through transcription of hepatocyte nuclear factor 1alpha which in turn can also affect the POMC response.

Role of HPF (Hibernation Promoting Factor) in Translational Activity in Escherichia coli

During the stationary phase of growth in Escherichia coli, ribosome modulation factor (RMF) and hibernation promoting factor (HPF) dimerize most 70S ribosomes to form 100S ribosomes. The process of 100S formation has been termed 'ribosomal hibernation'. Here, the contributions of HPF to 100S formation and translation were analysed in vitro. HPF bound to, but did not dimerize the 70S ribosome. RMF dimerized and formed immature 90S ribosomes. Binding of both HPF and RMF converted 90S ribosomes to mature 100S ribosomes, which is consistent with the in vivo data. The role of HPF in in vitro translation also was investigated. In an artificial mRNA poly (U)-dependent phenylalanine incorporation assay, HPF bound to ribosomal particles and inhibited translation. In contrast, in a natural MS2 mRNA-dependent leucine incorporation assay, bound HPF was removed and hardly inhibited normal translation. Multiple alignment and phylogenetic analyses indicates that the hibernation system mediated by the HPF homologue, RMF and 100S ribosome formation may be specific to the proteobacteria gamma group. In contrast, most bacteria have at least one HPF homologue, and these homologues can be classified into three types, long HPF, short HPF and YfiA.

Fundamental differences in bioaffinity of amino acid dehydrogenases for N 6- and S 6-linked immobilized cofactors using kinetic-based enzyme-capture strategies

Five different immobilized NAD + derivatives were employed to compare the behavior of four amino acid dehydrogenases chromatographed using kinetic-based enzyme capture strategies (KBECS): S 6-, N 6-, N 1-, 8′-azo-, and pyrophosphate-linked immobilized NAD +. The amino acid dehydrogenases were NAD +-dependent phenylalanine (EC 1.4.1.20), alanine (EC 1.4.1.1), and leucine (EC 1.4.1.9) dehydrogenases from various microbial species and NAD(P) +-dependent glutamate dehydrogenase from bovine liver (GDH; EC 1.4.1.3). KBECS for bovine heart l-lactate dehydrogenase (EC 1.1.1.27) and yeast alcohol dehydrogenase (EC 1.1.1.1) were also applied to assist in a preliminary assessment of the immobilized cofactor derivatives. Results confirm that the majority of the enzymes studied retained affinity for NAD + immobilized through an N 6 linkage, as opposed to an N 1 linkage, replacement of the nitrogen with sulfur to produce an S 6 linkage, or attachment of the cofactor through the C8 position or the pyrophosphate group of the cofactor. The one exception to this was the dual-cofactor-specific GDH from bovine liver, which showed no affinity for N 6-linked NAD + but was biospecifically adsorbed to S 6-linked NAD + derivatives in the presence of its soluble KBEC ligand. The molecular basis for this is discussed together with the implications for future development and application of KBECS.

Characterisation and cloning of a Na +-dependent broad-specificity neutral amino acid transporter from NBL-1 cells: a novel member of the ASC/B 0 transporter family

Na +-dependent neutral amino acid transport into the bovine renal epithelial cell line NBL-1 is catalysed by a broad-specificity transporter originally termed System B 0. This transporter is shown to differ in specificity from the B 0 transporter cloned from JAR cells [J. Biol. Chem. 271 (1996) 18657] in that it interacts much more strongly with phenylalanine. Using probes designed to conserved transmembrane regions of the ASC/B 0 transporter family we have isolated a cDNA encoding the NBL-1 cell System B 0 transporter. When expressed in Xenopus oocytes the clone catalysed Na +-dependent alanine uptake which was inhibited by glutamine, leucine and phenylalanine. However, the clone did not catalyse Na +-dependent phenylalanine transport, again as in NBL-1 cells. The clone encoded a protein of 539 amino acids; the predicted transmembrane domains were almost identical in sequence to those of the other members of the B 0/ASC transporter family. Comparison of the sequences of NBL-1 and JAR cell transporters showed some differences near the N-terminus, C-terminus and in the loop between helices 3 and 4. The NBL-1 B 0 transporter is not the same as the renal brush border membrane transporter since it does not transport phenylalanine. Differences in specificity in this protein family arise from relatively small differences in amino acid sequence.

Synthesis of a highly substituted N(6)-linked immobilized NAD(+) derivative using a rapid solid-phase modular approach: suitability for use with the kinetic locking-on tactic for bioaffinity purification of NAD(+)-dependent dehydrogenases.

This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases. Specifically, the synthesis of highly substituted N(6)-linked immobilized NAD(+) derivatives is described using a rapid solid-phase modular approach. Other modifications of the N(6)-linked immobilized NAD(+) derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 A) in an attempt to minimize nonbiospecific interactions. Analysis of the N(6)-linked NAD(+) derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S(6)-linked immobilized NAD(+) derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart l-lactate dehydrogenase (l-LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. l-phenylalanine dehydrogenase (l-PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and d-phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD(+)-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S(6)-linked immobilized cofactor derivatives than for the new N(6)-linked derivatives. In contrast, the NAD(+)-dependent phenylalanine dehydrogenase showed no affinity for the S(6)-linked immobilized NAD(+) derivative, but was locked-on strongly to the N(6)-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N(6)-linked immobilized NADP(+) derivative in the presence of d-phenylalanine. This differential locking-on of NAD(+)-dependent dehydrogenases to N(6)-linked and S(6)-linked immobilized NAD(+) derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD(+) immobilized via N(6)-linkage as compared to thiol-linkage.

Crystallization of NAD+-dependent phenylalanine dehydrogenase from Nocardia sp239.

The NAD+-dependent phenylalanine dehydrogenase from Nocardia sp239 has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. Two crystal forms were obtained in the presence and absence of the enzyme substrates phenylpyruvic acid or phenylalanine and its coenzyme NADH. Crystals of the native protein belong to the hexagonal system, with the space group being one of the enantiomorphic pair P6122 or P6522. The cell dimensions are a = b = 111.0, c = 174.5 A, alpha = beta = 90 and gamma = 120 degrees. Crystals grown from the protein co-crystallized with its substrates all belong to the trigonal system, space group P3121 or P3221, with unit-cell dimensions of a = b = 88.1, c = 112.6 A, alpha = beta = 90 and gamma = 120 degrees. Preliminary protein-sequencing experiments have established that this enzyme is related to the octameric PheDH's which are members of the wider superfamily of amino-acid dehydrogenases. However, gel-filtration studies suggest that this enzyme is active as a monomer. The full determination of the three-dimensional structure of this phenylalanine dehydrogenase will add to the understanding of the molecular basis of the differential substrate specificity within this enzyme superfamily. In turn this will contribute to the rational design of an amino-acid dehydrogenase which could be used for the diagnosis of phenylketonuria and for the chiral synthesis of high-value pharmaceuticals.

Thermostable phenylalanine dehydrogenase from a mesophilic microbacterium sp. strain DM 86-1

Bacteria that produced NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.20) were selected among l-methionine utilizers isolated from soil. A bacterial strain showing phenylalanine dehydrogenase activity was chosen and classified in the genus Microbacterium. Phenylalanine dehydrogenase was purified from the crude extract of Microbacterium sp. strain DM 86-1 (TPU 3592) to homogeneity as judged by SDS-polyacrylamide disc gel electrophoresis. The enzyme has an isoelectric point of 5.8 and a relative molecular weight (Mr) of approximately 330,000. The enzyme is composed of eight identical subunits with an Mr of approximately 41,000. The apparent Km values for l-phenylalanine and NAD+ were calculated to be 0.10 mM and 0.20 mM, respectively. No loss of the enzyme activity was observed upon incubation at 55 degrees C for 10 min.

ChemInform Abstract: Development of New Microbial Enzymes and Their Use in Organic Synthesis

New enzymes screened for specific conversions have been purified and characterized from various microorganisms, and successfully applied to organic synthesis. These include : 1) nitrile hydratase from Arthrobacter sp. and Pseudomonas chlororaphis, useful in the hydration of nitriles to produce amides, 2) NAD+-dependent phenylalanine dehydrogenase from Sporosarcina ureae and Bacillus sphaericus, useful in the reductive amination of phenylpyruvate to give L-phenylalanine, 3) D-aminpeptidase from Achromobacter sp. applicable to the kinetic resolution of amino acid amides to give D-amino acid, and the D-stereospecific aminolysis reaction in organic solvents of amino acid esters or amides to yield D-amino acid N-alkylamides.

Polyamines of Sulfur-Dependent Archaebacteria and Their Role in Protein Synthesis1

Several strains of the sulfur-dependent archaebacterium, Sulfolobus, were analyzed for their polyamine content. Caldine (norspermidine), spermidine, and thermine were found to be major components in all of the cells tested. The most abundant polyamine in all cultures examined was spermidine. The Langworthy strain had the highest spermine content, whereas S. acidocaldarius strain no. 7 was devoid of this polyamine. Cultures of strain no. 7 grown at 70 degrees C were rich in spermidine and caldine (triamines) and the thermine: spermidine ratio was much lower than that of cultures grown at 78 degrees C. Equal amounts of thermine and spermidine were present in strain DSM 1616. Preincubation of Langworthy strain extracts at 10 degrees C did not overcome the requirement for polyamines in protein synthesis. Putrescine exerted a concentration-dependent inhibition of the spermine-induced stimulation of protein synthesis at 70 degrees C. Increasing concentrations (6 and 9 mM) of spermine and thermine progressively inhibited poly(U)-dependent phenylalanine incorporation at 45 degrees C to about the same extent, whereas the same concentrations of these polyamines had little effect on the reaction at 70 degrees C. Although 3 mM spermine had only a slight stimulatory effect on the attachment of phenylalanine to tRNA at 65 degrees C, this polyamine had a pronounced effect on the formation of 70S ribosomes in a standard buffer containing 10 mM Mg2+. Increasing the Mg2+ concentration to 30 mM in the absence of spermine was even more effective in causing the reassociation of subunits to form 70S particles.

Neither barium nor calcium prevents the inhibition by Bacillus thuringiensis δ‐endotoxin of sodium‐ or potassium gradient‐dependent amino acid accumulation by tobacco hornworm midgut brush border membrane vesicles

AbstractRapid filtration assays were used to determine the effects of barium, calcium and an insecticidal δ‐endotoxin from Bacillus thuringiensis on sodium and potassium ion gradient dependent phenylalanine accumulation by brush border membrane vescles from the larval midgut of the tobacco hornworm (Manduca sexta). Neither barium nor calcium had a significant effect on sodium ion gradient dependent phenylalanine accumulation by the membrane vesicles. Both barium and calcium inhibited potassium ion gradient dependent phenylalanine accumulation by the membrane vesicles. B. thuringiensis δ‐endotoxin inhibited both sodium and potassium ion gradient dependent phenylalanine accumulation by the vesicles. Inhibition of both sodium and potassium ion gradient dependent phenylalanine accumulation increased similarly with increasing δ‐endotoxin inhibition of either sodium or potassium dependent phenylalanine accumulation by the vesicles.

新規微生物酵素の開発と有機合成への利用

New enzymes screened for specific conversions have been purified and characterized from various microorganisms, and successfully applied to organic synthesis. These include : 1) nitrile hydratase from Arthrobacter sp. and Pseudomonas chlororaphis, useful in the hydration of nitriles to produce amides, 2) NAD+-dependent phenylalanine dehydrogenase from Sporosarcina ureae and Bacillus sphaericus, useful in the reductive amination of phenylpyruvate to give L-phenylalanine, 3) D-aminpeptidase from Achromobacter sp. applicable to the kinetic resolution of amino acid amides to give D-amino acid, and the D-stereospecific aminolysis reaction in organic solvents of amino acid esters or amides to yield D-amino acid N-alkylamides.

Purification and characterization of a dimeric phenylalanine dehydrogenase from Rhodococcus maris K-18

NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.) was purified to homogeneity from a crude extract of Rhodococcus maris K-18 isolated from soil. The enzyme had a molecular mass of about 70,000 daltons and consisted of two identical subunits. The enzyme catalyzed the oxidative deamination of L-phenylalanine and several other L-amino acids and the reductive amination of phenylpyruvate and p-hydroxyphenylpyruvate. The enzyme required NAD+ as a natural coenzyme. The NAD+ analog 3-acetylpyridine-NAD+ showed much greater coenzyme activity than did NAD+. D-Phenylalanine, D-tyrosine, and phenylethylamine inhibited the oxidative deamination of L-phenylalanine. The enzyme reaction was inhibited by p-chloromercuribenzoate and HgCl2. Initial-velocity and product inhibition studies showed that the reductive amination proceeded through a sequential ordered ternary-binary mechanism. NADH bound first to the enzyme, followed by phenylpyruvate and then ammonia, and the products were released in the order L-phenylalanine and NAD+. The Michaelis constants were as follows: L-phenylalanine, 3.8 mM; NAD+, 0.25 mM; NADH, 43 microM; phenylpyruvate, 0.50 mM; and ammonia, 70 mM.

Novel phenylalanine dehydrogenases from Sporosarcina ureae and Bacillus sphaericus. Purification and characterization.

NAD+-dependent phenylalanine dehydrogenases were purified 1,500- and 1,600-fold, and crystallized from Sporosarcina ureae SCRC-R04 and Bacillus sphaericus SCRC-R79a, respectively. The purified enzymes were homogeneous as judged by disc gel electrophoresis. The enzyme from S. ureae has a molecular weight of 305,000, while that of B. sphaericus has a molecular weight of 340,000. Each is probably composed of eight subunits identical in molecular weight. The S. ureae enzyme showed a high substrate specificity in the oxidative deamination reaction acting on L-phenylalanine, while that of B. sphaericus acted on L-phenylalanine and L-tyrosine. The enzymes had lower substrate specificities in the reductive amination reaction acting on alpha-keto acids. The Sporosarcina enzyme acted on phenylpyruvate, alpha-ketocaproate, alpha-keto-gamma-methylthiobutyrate and rho-hydroxyphenylpyruvate. The Bacillus enzyme acted on rho-hydroxyphenylpyruvate, phenylpyruvate, and alpha-keto-gamma-methylthiobutyrate. The enzyme from B. sphaericus catalyzes The enzyme from B. sphaericus catalyzes the transfer of pro-S (B) hydrogen from NADH.

Effect of norepinephrine on renal tubular NaKATPase and oxygen consumption

Previous studies from this laboratory have demonstrated that norepinephrine (NE) increases sodium dependent phenylalanine uptake by in vitro rat cortical tubules. In the present study, we have examined whether the observed increase in proximal tubular sodium transport, during NE treatment, is related to changes in membrane NaKATPase and cellular oxygen consumption. Treatment of intact tubules with NE increased microsomal NaKATPase activity but had no effect on cellular oxygen consumption or ouabain inhibitable oxygen consumption. The increased NaKATPase activity is consistent with the observed increase in sodium transport while the lack of a detectable effect on oxygen consumption suggests that the increased transport does not require additional oxygen utilization.

Renal transport of neutral amino acids. Tubular localization of Na+-dependent phenylalanine- and glucose-transport systems.

The transport properties for phenylalanine and glucose in luminal-membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit kidney were studied by a spectrophotometric method. Uptake of phenylalanine as well as of glucose by the two types of membrane vesicles was found to be Na+-dependent, electrogenic and stereospecific. Na+-dependent transport of L-phenylalanine by outer-cortical membrane vesicles could be accounted for by one transport system (KA congruent to 1.5 mM). By contrast, in the outer-medullary preparation, L-phenylalanine transport occurred via two transport systems, namely a high-affinity system with K1A congruent to 0.33 mM and a low-affinity system with K2A congruent to 7 mM respectively. Na+-dependent uptake of D-glucose by pars convoluta and pars recta membrane vesicles could be described by single, but different, transport systems, namely a low-affinity system with KA congruent to 3.5 mM and a high-affinity system with KA congruent to 0.30 mM respectively. Attempts to calculate the stoichiometry of the different Na+/D-glucose transport systems by using Hill-type plots revealed that the ratio of the Na+/hexose co-transport probably is 1:1 in the case of pars convoluta and 2:1 in membrane vesicles from pars recta. The Na+/L-phenylalanine stoichiometry of the pars convoluta transporter probably is 1:1. Both the high-affinity and the low-affinity Na+-dependent L-phenylalanine transport system of pars recta membrane vesicles seem to operate with a 1:1 stoichiometry. The physiological importance of the arrangement of low-affinity and high-affinity transport systems along the kidney proximal tubule is discussed.

Translational apparatus in relation to the naturally occurring stoppage of protein synthesis in the ovaries of Bombyx mori

1. 1. Some parameters for protein synthesis were investigated with the developing ovaries of Bombyx mori. 2. 2. The cell-free translational activities of cytoplasmic RNA fluctuated as expected from the reported patterns for the bulk chorion protein synthesis and fell rapidly at the later period of ovarian development. 3. 3. This fall was not accompanied by changes in content of ribosomes and activity of S-100 enzymes but by a decrease in activity of ribosomes as assayed by a poly(U)-dependent phenylalanine system at a low magnesium ion concentration.

K +-dependent phenylalanine uptake in membrane vesicels isolated from the midgut of Philosamia cynthia larvae

Membrane vesicles prepared from the midguts of Philosamia cynthia larvae (Lepidoptera) show a concentrative uptake of phenylalanine in the presence of salt gradients. Unlike mammalian intestines, the highest accumulation of the amino acid occurs with a potassium salt gradient. Glucose is very poorly permeable across the vesicles.

Cycloheximide resistance can be mediated through either ribosomal subunit.

Two cycloheximide-resistant mutants of Tetrahymena thermophila were analyzed to determine the site of their cycloheximide resistance. The mutations in both strains had been previously shown to be genetically dominant and located at separate loci (denoted Chx-A and Chx-B). Strains carrying these mutations were readily distinguished by the extent to which they were resistant to the drug. The homozygous double mutant was more resistant than either single mutant. Cell-free extracts of wild type and of the three mutant strains, assayed for protein synthetic activity by both runoff of natural mRNA and poly(U)-dependent phenylalanine polymerization, demonstrated that in vitro the mutants were all more resistant than the wild type. Further fractionation of the cell-free systems into ribosomes and supernates localized cycloheximide resistance to the ribosome for both Chx-A and Chx-B homozygotes. Ribosome dissociation and pairwise subunit mixing in the in vitro system indicated that ribosome resistance was conferred by the 60S subunit from one strain whereas resistance in the other strain was mediated through the 40S subunit. This was further confirmed by reconstruction of all four cycloheximide-resistance "phenotypes" by mixing ribosomal subunits from appropriate strains. This finding suggests that the mechanisms by which these mutations confer resistance to cycloheximide are different.

Decreased activity of peptide-elongation factors after treatment with cholesterol esterase

1. Peptide-elongation factors were purified from rat liver and treated with cholesterol esterase and phospholipase A2 immobilized on Sepharose 4B. 2. Binding of L-[3H]-phenylalanyl-tRNA to 40S ribosomal subunits was decreased by approx. 70% and to polyribosomes by 30% in the presence of the binding factor incubated with cholesterol esterase. Treatment of this factor with immobilized phospholipase A2 decreased the binding to smaller ribosomal subunits by only about 15%. 3. Poly(U)-dependent phenylalanine polymerization by ribosomal subunits was decreased to approx. 30% of its original value by treatment of both elongation factors with cholesterol esterase. 4. The normal activity of esterase-treated elongation factor in both the binding reaction and peptide-elongation assay was fully recovered by the addition of cholesteryl 14-methyl-hexadecanoate. 5. Different classes of lipids present in peptide-elongation factor 1 have apparently different functions. Whereas phospholipids are required to maintain the strcture of heavy aggregates of this factor, the presence of cholesteryl 14-methylhexadecanoate is obviously necessary for the normal function of peptide-elongation factors.