Search

Protein synthesis requires the pairing of amino acids with tRNAs catalyzed by the aminoacyl-tRNA synthetases. The synthetases are highly specific, but errors in amino acid selection are occasionally made, opening the door to inaccurate translation of the genetic code. The fidelity of protein synthesis is maintained by the editing activities of synthetases, which remove noncognate amino acids from tRNAs before they are delivered to the ribosome. Although editing has been described in numerous synthetases, the reaction mechanism is unknown. To define the mechanism of editing, phenylalanyl-tRNA synthetase was used to investigate different models for hydrolysis of the noncognate product Tyr-[tRNA.sup.Phe]. Deprotonation of a water molecule by the highly conserved residue [beta]His-265, as proposed for threonyl-tRNA synthetase, was excluded because replacement of this and neighboring residues had little effect on editing activity. Model building suggested that, instead of directly catalyzing hydrolysis, the role of the editing site is to discriminate and properly position noncognate substrate for nucleophilic attack by water. in agreement with this model, replacement of certain editing site residues abolished substrate specificity but only reduced the catalytic efficiency of hydrolysis 2-to 10-fold. in contrast, substitution of the 3'-OH group of [tRNA.sup.Phe] severely impaired editing and revealed an essential function for this group in hydrolysis. The phenylalanyl-tRNA synthetase editing mechanism is also applicable to threonyl-tRNA synthetase and provides a paradigm for synthetase editing. proofreading | translation | phenylalanine

Heptameric pores formed in the endosomal membrane by the protective antigen moiety of anthrax toxin serve as portals for entry of the enzymatic moieties of the toxin into the cytosol. In the aqueous lumen of each pore is a 'Phe clamp,' a heptad of narrowly apposed Phe residues (Phe-427), that catalyzes the unfolding and translocation of the enzymatic moieties across the membrane. Here, we provide evidence for a 'loop swap' between neighboring protective antigen subunits, which is required for efficient translocation and is mediated by a salt bridge formed between the side chains of Lys-397 and Asp-426. We propose that the interaction between residues 397 and 426 creates a structural framework that positions Phe-427 within the pore lumen, forming a functional Phe clamp and, hence, a translocation-competent pore. intersubunit | translocation | planar lipid bilayer | lethal factor | protective antigen

An important phenylalanine-derived volatile compound produced by plants is 2-phenylethanol. It is a major contributor to flavor in many foods, including fresh fruits, such as tomato, and an insect-attracting scent in roses and many other flowers. Despite the centrality of 2-phenylethanol to flavor and fragrance, the plant genes responsible for its synthesis have not been identified. Here, we describe a biosynthetic pathway for 2-phenylethanol and other phenylalanine-derived volatiles in tomato fruits and a small family of decarboxylases (LeAADC1A, LeAADC1B, and LeAADC2) that can mediate that pathway's first step. These enzymes each catalyze conversion of phenylalanine to phenethylamine and tyrosine to tyramine. Although tyrosine is the preferred substrate in vitro, phenylalanine levels in tomato fruits far exceed those of tyrosine, indicating that phenylalanine is a physiological substrate. Consistent with this view, overexpression of either LeAADCIA or LeAADC2 in transgenic tomato plants resulted in fruits with up to 10-fold increased emissions of the products of the pathway, including 2-phenylacetaldehyde, 2-phenylethanol, and 1-nitro-2phenylethane. Further, antisense reduction of LeAADC2 significantly reduced emissions of these volatiles. Besides establishing a biosynthetic route, these results show that it is possible to change phenylalanine-based flavor and aroma volatiles in plants by manipulating expression of a single gene. metabolic engineering | phenylalanine | taste

The shikimate pathway resulting in three aromatic amino acids is initiated in different organisms by two and three 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases, respectively. Aro3p and Aro4p are the yeast enzymes feedback-inhibited by phenylalanine and tyrosine, respectively. A yeast strain deficient in the general control transcriptional regulatory system of amino acid biosynthesis is unable to live in the presence of high amounts of phenylalanine and tyrosine. Here, we show that this yeast strain can be rescued by the expression of aroH from Escherichia coil encoding the tryptophan-regulated AroH as third isoenzyme. Yeast carrying Ec AroH as the only enzyme for the initial step of the shikimate pathway can grow in the absence of tryptophan. Without aromatic amino acids, this yeast strain survives only when the yeast ARO3 promoter instead of the AR04 promoter drives E. coil aroH. The detailed analysis of Aro3p and Aro4p revealed a triple feedback control by tyrosine/phenylalanine and tryptophan. Dissecting this control allowed engineering of Aro4p S195A as an enzyme, which is inhibited like AroH only by tryptophan. In addition, Aro4p variants were constructed that show an equally strong inhibition by tyrosine and tryptophan (Aro4p P165G Q302R) and in which the regulation by tyrosine and tryptophan was reversed (Aro4p P165G). Our data suggest that yeast possesses only two instead of three isogenes encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases because both isoenzymes can be fine tuned by tryptophan as additional effector and because transcriptional regulation by the general control system can be induced as backup when aromatic amino acids in the environment are imbalanced. general control | Aro4p | AroH

Byline: Yoshihiro Ozeki (1), Yukie Chikagawa (1), Souichi Kimura (1), Hyun-cheol Soh (1), Kazuhiro Maeda (1), Wannarat Pornsiriwong (1), Masayuki Kato (1), Hirofumi Akimoto (1), Mikiko Oyanagi (1), Takashi Fukuda (1), Takatoshi Koda (2), Yoshio Itoh (1), Akiyo Yamada (1), Eric Davies (3), Hiroshi Ueno (4), Junko Takeda (4) Keywords: A-box Carrot 2,4-Dichlorophenoxyacetic acid L-box P-box Phenylalanine ammonia-lyase Abstract: Deletion mutants of the carrot phenylalanine ammonia-lyase gene promoter were used to survey cis-elements for their effect on expression of promoter activity by transient expression. Two putative cis-elements were required to give full activity, but a third might be the most important in regulation of the promoter by 2,4-dichlorophenoxyacetic acid. Author Affiliation: (1) Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan (2) , San-Ei Gen F.F.I., Inc., Osaka, Japan (3) Botany Department, North Carolina State University, Raleigh, North Carolina, USA (4) Department of Food Science and Nutrition, Nara Women's University, Nara, Japan Article History: Received Date: 24/09/2002 Accepted Date: 18/12/2002 Article note: Electronic Publication

Byline: Yoshihiro Ozeki (1), Yoshio Ito (1), Nobuhiro Sasaki (1), Mikiko Oyanagi (1), Hirofumi Akimoto (1), Yukie Chikagawa (1), Junko Takeda (2) Keywords: Keywords: Anthocyanin, Carrot, Differentiation, myb, Phenylalanine ammonia-lyase (PAL) Abstract: and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed. Author Affiliation: (1) Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184--8588 Japan, JP (2) Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606--8502 Japan, JP Article note: Received 10 June 2000/ Accepted in revised form 1 July 2000

The derivation of a quantitative model of phenylalanine metabolism in humans is described. The model is based on the kinetic properties of pure recombinant human phenylalanine hydroxylase and on estimates of the in vivo rates of phenylalanine transamination and protein degradation. Calculated values for the steady-state concentration of blood phenylalanine, rate of clearance of phenylalanine from the blood after an oral load of the amino acid, and dietary tolerance of phenylalanine all agree well with data from normal as well as from phenylketonuric patients and obligate heterozygotes. These calculated values may help in the decision about the degree of restriction of phenylalanine intake that is necessary to achieve a satisfactory clinical outcome in classical patients and in those with milder forms of the disease.

Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 4.3.1.5) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 1.14.16.1), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N[prime]-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA) - both pharmacologic (with a clear dose-response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease.

Byline: LF Pereira (1), PH Goodwin (2), L Erickson (3) Keywords: Keywords: Gene expression, Manihot esculenta, Phenylalanine ammonia lyase, Xanthomonas axonopodis pv. manihotis, Xanthomonas cassavae Abstract: ). PAL enzyme activity and MEPAL mRNA levels were measured in leaves of cultivar MCOL 22 following mechanical wounding or inoculation with 2 bacterial pathogens. MCOL 22 is resistant to Xanthomonas cassavae, which causes cassava bacterial necrosis and susceptible to X. axonopodis pv. manihotis, which causes cassava bacterial blight. PAL enzyme activity in the resistant interaction was significantly higher than in the susceptible interaction or the control. Similarly, MEPAL transcripts were detected in leaves during the resistant interaction, but not during the susceptible reaction. RFLP analysis with MEPAL revealed a divergence between South American and African/Asian cultivars and showed promise for MEPAL as a marker for resistance to Xanthomonads. Author Affiliation: (1) Area de Ecofisiologia, IAPAR, Londrina, Pr Brasil, BR (2) Department of Environmental Biology, University of Guelph, Guelph Ontario NIG 2WI, Canada, CA (3) Department of Plant Agriculture, University of Guelph, Guelph Ontario NIG 2WI, Canada, CA Article note: Received 25 September 1998/ Accepted in revised form 4 January 1999

Phenylalanine hydroxylase stimulator (PHS) is a component of the phenylalanine hydroxylation system that is involved in the regeneration of the cofactor tetrahydrobiopterin. It is also identical to the dimerization cofactor of hepatocyte nuclear factor 1 (HNF1) (DCoH) that is able to enhance the transcriptional activity of HNF1. Moreover, it has the structural potential for binding macromolecules such as proteins and nucleic acids, consistent with its involvement in gene expression. We investigated whether PHS/DCoH could enhance the expression of phenylalanine hydroxylase (PAH). Cotransfection assays showed that DCoH itself could not transactivate the 9-kb human PAH 5[prime] flanking fragment. However, this 9-kb fragment was transactivated by HNF1 in a dose-dependent manner with a maximum of nearly 8-fold activation; DCoH potentiated this transactivation by another 1.6-fold. The HNF1 binding sites were located at -3.5 kb in a region that is 77.5% identical to the mouse liver-specific hormone-inducible PAH gene enhancer. This study suggests a possible dual function of PHS in vivo in the human phenylalanine hydroxylation system: it is involved in the regeneration of the cofactor tetrahydrobiopterin and can also enhance the expression of the human PAH gene.

A study was conducted to characterize the forms of phenylalanine hydroxylase-stimulating protein/DCoH found in patients with hyperphenylalanemia and to compare them with the wild-type enzyme in the GenBank. Gene cloning revealed that mutant C82R is similar to the wild-type enzyme in that both are heat stable and forms a tetramer. However, C82R is more susceptible to proteolysis and are expressed in lower levels in transfected mammalian cells than the wild-type protein.

The mechanism of reaction of the phenylalanine ammonia lyase (PAL) involves a transient Fridel-Crafts-like acylation of the phenyl ring in which the crucial step is an electrophilic attack of the prosthetic group. Consequently a carbenium ion is formed in which the beta-Hsi atom is activated in a similar way as it is in the nitro analogue. The catalytic cycle is completed by the elimination of ammonia and the restoration of the aromatic ring and the prosthetic group.

NMR spectroscopic and assay studies of the structure and function of the carrier protein family of Ras proteins indicate the presence of a conserved phenylalanine-156 (F156) residue to be essential for maintaining the biological function and structure of Ras proteins. An F156 mutation in the wild-type Ras enhances the guanine nucleotide production, stimulated Ras transforming potential and affected the protein structure by disrupting the alpha-helix and beta-sheets.

The alkali metal cations [Na.sup.+] and [K.sup.+] have several important physiological roles, including modulating enzyme activity. Recent work has suggested that alkali metal cations may be coordinated by [Pi] systems, such as the aromatic amino acid side chains. The ability of [K.sup.+] to interact with an aromatic ring has been assessed by preparing a family of synthetic receptors that incorporate the aromatic side chains of phenylalanine, tyrosine, and tryptophan. These receptors are constructed around a diaza-18-crown-6 scaffold, which serves as the primary binding site for an alkali metal cation. The ability of the aromatic rings to coordinate a cation was determined by crystallizing each of the receptors in the presence of [K.sup.+] and by solving the solid state structures. In all cases, complexation of [K.sup.+] by the [Pi] system was observed. When possible, the structures of the unbound receptors also were determined for comparison. Further proof that the aromatic ring makes an energetically favorable interaction with the cation was obtained by preparing a receptor in which the arene was perfluorinated. Fluorination of the arene reverses the electrostatics, but the aromaticity is maintained. The fluorinated arene rings do not coordinate the cation in the solid state structure of the [K.sup.+] complex. Thus, the results of the predicted electrostatic reversal were confirmed. Finally, the biological implications of the alkali metal cation-[Pi] interaction are addressed.