Rapid automated ion-exchange analysis of plasma tyrosine and phenylalanine with data print-out

A rapid automated method with data print-out is described for quantitation of plasma phenylalanine and tyrosine from 0.100 ml of sample. The system uses the Rank-Hilger Chromaspek amino acid analyser linked to a Digico M16E computer.Amino acid concentrations up to 3000 μM can be quantitated without repeat dilutions and assessment of precision at the 500 μM level, produced coefficients of variation of 2.2% for tyrosine and 2.5% for phenylalanine. Recovery determinations from a plasma pool gave a mean recovery of 99.4% for tyrosine and 99.7% for phenylalanine.Correlation with established fluorimetric techniques was excellent (r = 0.986 for tyrosine, r = 0.976 for phenylalanine). By using the same resin column for both the rapid separation of tyrosine and phenylalanine, and the standard physiological fluid separation, full analysis capability is retained with easy interchange between the two systems.     

Regulation of Aromatic Amino Acid Biosynthesis and Production of Tyrosine and Phenylalanine m Brevibacterium flavum

In Brevibacterium flavum, prephenate dehydratase in the phenylalanine specific biosynthetic pathway was strongly inhibited by phenylalanine and activated by tyrosine. Furthermore. the inhibition by phenylalanine was completely reversed by tyrosine. Inhibition by tyrosine of prephenate dehydrogenase in the tyrosine specific pathway was very weak. Overall regulation mechanism of the aromatic amino acid biosynthesis in B. flavum was proposed on the bases of these results and the previous findings on 3-deoxy-D-arabino-heptulosonate-7- phosphate synthetase(DAHP synthetase*) of the common pathway and on anthranilate synthetase of the tryptophan specific pathway. Two types of m-fluorophenylalanine(mFP) resistant mutants which accumulated phenylalanine alone or both phenylalanine and tyrosine, respectively, were derived. The accumulation in the former mutants was inhibited by tyrosine, but that in the latter was affected neither by tyrosine nor by phenylalanine. DAHP synthetase of the latter mutants had been desensitized from the synergistic feedback inhibition by tyrosine and phenylalanine, while prephenate dehydratase of the former mutants had been desensitized in the feedback inhibition by phenylalanine. Tyrosine auxotroph accumulated phenylalanine under tyrosine limitation and its accumulation was inhibited by the excessive addition of tyrosine. Phenylalanine auxotroph accumulated tyrosine under phenylalanine limitation and its accumulation was inhibited by the excessive addition of phenylalanine. These results in vivo strongly supported the proposed regulation mechanism in which synthesis of phenylalanine in preference to tyrosine was assumed.     

The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.Abbreviations DHPL 3,4-dihydroxyphenyllactate - DHPP 3,4-dihydroxyphenylpyruvate - pHPL 4-hydroxyphenyllactate - pHPP 4-hydroxyphenylpyruvate - RA rosmarinic acid     

Formation of Phenolic and Indolic Compounds by Anaerobic Bacteria in the Human Large Intestine

Abstract Batch culture incubations were used to investigate the effects of pH (6.8 or 5.5) and carbohydrate (starch) availability on dissimilatory aromatic amino acid metabolism in human fecal bacteria. During growth on peptide mixtures, tyrosine and phenylalanine fermentations occurred optimally at pH 6.8, while individual metabolic reactions were inhibited by up to 80% in the presence of 10 g l⁻¹ starch. Tryptophan metabolites were not detected in these experiments. When free amino acids replaced peptides, phenol production was increased during carbohydrate fermentation, although formation of p-cresol, another tyrosine metabolite was strongly inhibited. Phenylpropionate, which is produced from phenylalanine, was unaffected by starch. Tryptophan was fermented in these studies, although indole production was reduced in the starch fermentors. The importance of different fermentation substrates (casein, peptide mixtures, free amino acids) on aromatic amino acid metabolism was investigated in incubations of material taken from the proximal bowel. The phenylalanine metabolites, phenylacetate and phenylpropionate, were the principal phenolic compounds formed from all three substrates. Phenol was the major tyrosine metabolite produced in casein and peptide fermentations, while hydroxyphenylpropionate was a more important tyrosine product from free amino acids. Indole was the sole product of tryptophan metabolism, but was formed only from the free amino acid. Bacterial metabolism of individual phenolic and indolic compounds was also investigated. Phenol, p-cresol, phenylacetate, phenylpropionate, 4-ethylphenol, indole, indoleacetate, and indolepropionate were not metabolized by colonic bacteria. However, hydroxyphenylacetate was hydrolyzed to p-cresol, while hydroxyphenylpropionate was transformed into phenylpropionate. Indolepyruvate was either converted to indoleacetate or metabolized into indole. Indolepropionate, and to a lesser degree indoleacetate were produced from indolelactate. These data show that human colonic anaerobes are able to extensively degrade either free or peptide-bound aromatic amino acids, with the concomitant formation of toxic metabolic products. These processes are controlled to a significant degree by environmental factors such as pH and carbohydrate availability, and this ultimately influences the types and amounts of fermentation products that can be formed in different regions of the large bowel. Received: 25 January 1996; Accepted: 8 May 1996     

Rosmarinic acid formation and differential expression of tyrosine aminotransferase isoforms in Anchusa officinalis cell suspension cultures

Time-course changes in rosmarinic acid (RA) formation and activities of tyrosine aminotransferase (TAT) isoforms were examined in Anchusa officinalis suspension cultures. Three TAT isoforms (TAT-1, TAT-3, TAT-4) were resolved by Mono-Q anion-exchange column chromatography. The proportion of the TAT-3 activity within the total TAT activity remained high regardless of the growth stage of the cultured cells. TAT-1 activity was positively correlated with the rate of RA biosynthesis during linear growth stage of the culture cycle, while TAT-4 activity was rapidly induced in conjunction with transfer to fresh medium coincident with a transient increase in RA synthesis. Based on these results, as well as the substrate specificity of each TAT isoform, it was concluded that both TAT-1 and TAT-4 are closely involved in RA biosynthesis. TAT-1 controls conversion of tyrosine to 4-hydroxyphenyl pyruvate, and TAT-4 acts by participating in the formation of tyrosine and phenylalanine via prephenate.Abbreviations PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - RA rosmarinic acid     

Intracellular concentrations of phenylalanine,tyrosine and α-aminobutyric acid in 13 homozybotes and 19 heterozygotes for phenylketonuria (PKU) compared with 26 normals

Summary Intracellular concentrations of phenylalanine, tyrosine, -aminobutyric acid, and seven other aminoacids (glycine, alanine, valine, cystine, methionine, isoleucine, leucine) were measured in lymphocytes of 13 homozygotes and 19 heterozygotes for phenylketonuria and in lymphocytes of 26 normals. Intracellular concentrations for phenylalanine, tyrosine, and -aminobutyric acid were significantly higher in homo- and heterozygotes than in normals (P<0.001; P<0.01). For the other seven aminoacids there were no or only questionable differences. Between homo-and heterozygotes there was no difference in any of the aminoacids. The intracellular phenylalanine: tyrosine ratio was essentially the same in all three groups of individuals. There was no correlation between intracellular phenylalanine above or below 10nmol/10⁶ cells and IQ in heterozygotes. The same is true for phenylalanine: tyrosine ratio greater or smaller than 1. In homozygotes there was no correlation between intracellular phenylalanine and age—to which DQ/IQ is correlated. There was no significant difference in intracellular phenylalanine between homozygotes with blood levels above and below 908 mol/l (15 mg/100 ml) at the time of blood sampling and no correlation between intra- and extracellular phenylalanine concentrations.Among the 26 normals there were only two with intracellular phenylalanine above 10 nmol/10⁶ cells, both showing phenylalanine loading test curves suggestive of heterozygosity.The results are discussed and important functions of the cell wall are proposed. The formation of an abnormal unknown intracellular metabolite being the real noxious agent could explain the incomparably different degrees of brain dysfunction in individuals with equal though elevated intracellular phenylalanine concentrations, i.e., homozygotes and heterozygotes for PKU.     

Biochemical Evaluation of the Decarboxylation and Decarboxylation-Deamination Activities of Plant Aromatic Amino Acid Decarboxylases

Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site residues.     

Physiological role of tyrosine transport systems in Halobacterium salinarium

The quantitative content of three transport systems for aromatic amino acids in cells of Halobacterium salinarium was measured: the common system (K _m is about 10^-6 M) and two tyrosine-specific systems with high and low affinity (K _m is about 10^-8 and 10^-5 M, respectively). To determine the activity of each of three systems separately, a method was developed based on the selective phenylalanine effect on these activities. When phenylalanine exeeds [¹⁴C]tyrosine by four to sixforld, it inhibits competitively the activity of the common system, and its 50- to 100-fold molar excess is inhibitory in a non-competitive way for the specific high affinity system (HAT system). The specific low affinity system (LAT system) is practically insensitive to phenylalanine. The activities of tyrosine-specific transport systems are slightly dependent on the culture age, and the observed decrease in transport activity during growth is due mainly to the decreased content of the common system. The HAT system formation is regulated by the repression type, and the effectors are aromatic amino acids especially tyrosine itself. The physiological sense of the tyrosine transport system's multiplicity in H. salinarium is discussed.     

Breeding of Phenylalanine-producing Brevibacterium flavum Strains by Removing Feedback Regulation of Both the Two Key Enzymes in Its Biosynthesis

The growth of a mFP-resistant Brevibacterium flavum mutant, No. 221-43, having PDT^R was synergistically and completely inhibited by mFP plus Tyr-Glu, but not by mFP plus tyrosine or pFP plus Tyr-Glu, whereas that of a mutant having was only partially inhibited by mFP plus Tyr-Glu. Tyr-Glu could replace tyrosine required for the growth of a tyrosine auxotroph. The phenylalanine uptake was competitively inhibited by tyrosine and the tyrosine uptake by phenylalanine. The phenylalanine uptake was also inhibited by mFP, but not by Tyr-Glu. Mutants having both PDT^R and DS^R derived from strain No. 221-43 were effectively selected by the resistance to mFP plus Tyr-Glu, and produced much larger amounts of phenylalanine, with small amounts of tyrosine, than the parent. By the same method, mutants having DS^R and PDT^R, which produced 23.4 g/l of phenylalanine at maximum, were obtained from a pFP-resistant tyrosine auxotroph having PDT^R which produced 18 g/l. Similar mutants were also obtained from a tryptophan-producing strain, but produced smaller amounts of tryptophan than the parent, whereas the total amounts of tryptophan and phenylalanine produced were increased.     

Expression of bacterial tyrosine ammonia-lyase creates a novel p-coumaric acid pathway in the biosynthesis of phenylpropanoids in Arabidopsis

Some flavonoids are considered as beneficial compounds because they exhibit anticancer or antioxidant activity. In higher plants, flavonoids are secondary metabolites that are derived from phenylpropanoid biosynthetic pathway. A large number of phenylpropanoids are generated from p-coumaric acid, which is a derivative of the primary metabolite, phenylalanine. The first two steps in the phenylpropanoid biosynthetic pathway are catalyzed by phenylalanine ammonia-lyase and cinnamate 4-hydroxylase, and the coupling of these two enzymes forms a rate-limiting step in the pathway. For the generation of p-coumaric acid, the conversion from phenylalanine to p-coumaric acid that is catalyzed by two enzymes can be theoretically performed by a single enzyme, tyrosine ammonia-lyase (TAL) that catalyzes the conversion of tyrosine to p-coumaric acid in certain bacteria. To modify the p-coumaric acid pathway in plants, we isolated a gene encoding TAL from a photosynthetic bacterium, Rhodobacter sphaeroides, and introduced the gene (RsTAL) in Arabidopsis thaliana. Analysis of metabolites revealed that the ectopic over-expression of RsTAL leads to higher accumulation of anthocyanins in transgenic 5-day-old seedlings. On the other hand, 21-day-old seedlings of plants expressing RsTAL showed accumulation of higher amount of quercetin glycosides, sinapoyl and p-coumaroyl derivatives than control. These results indicate that ectopic expression of the RsTAL gene in Arabidopsis enhanced the metabolic flux into the phenylpropanoid pathway and resulted in increased accumulation of flavonoids and phenylpropanoids.     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Amino acid decarboxylases in some fungi

The authors describe a method for the determination of decarboxylase activity in fungi. Some of the strains of test fungi (Stachybotrys alternans, Fusarium andAspergillus) displayed arginine, lysine, phenylalanine, asparaginic and glutamic decarboxylase activity. No tyrosine, tryptophane, histidine or ornithine decarboxylase activity was found. Some of the factors influencing the activity of these enzymes are discussed.     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain

The incorporation of [³H]phenylalanine, [³H]tyrosine, and [³H]tryptophan into protein and amino acyl–tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl–tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl–tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl–tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both K_m and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than K_m.     

Mechanism of irreversible inactivation of phenylalanine-4- and tryptophan-5-hydroxylases by [4-36Cl, 2-14C]p-chlorophenylalanine: A revision

Intraperitoneal injection of [4-³⁶Cl, 2-¹⁴C]p-chlorophenylalanine (pCPA) (300 mg/kg) in rats revealed absence of chlorine in pure hepatic phenylalanine hydroxyase, while the carbon label appeared as 1–4 moles/mole of [¹⁴C]tyrosine in the inactivated phenylalanine and cerebral tryptophan-5-hydroxylase. Crystalline muscle aldolase and tyrosine hydroxylase also revealed the presence of [2-¹⁴C]tyrosine from [2-¹⁴C]pCPA without inactivating these enzymes. Injection of L-[(U)-¹⁴C] tyrosine led to its incorporation into the above enzymes, but to a different degree without altering the enzyme activity. Repeated injections ofp-chlorophenylacetic acid had no effect on phenylalanine or tryptophan-hydroxylase. Administration of pCPA did not change the levels of cerebral biopterins. Reexamination of the effect of cycloheximide on reversing enzymic inactivation by pCPA failed to confirm our earlier observation.     

Control of Aromatic Amino Acid Biosynthesis in Cultured Plant Tissues: Effect of Intermediates and Aromatic Amino Acids on Free Levels

Shikimate, anthranilate, indole, l -tryptophan, phenylpyruvate, l -p henylalanine, p-hydroxyphenylpyruvate or l -tyrosine were added to suspension-cultured Nicotiana tabacum (tabacco) and Daucus carota (carrot) tissues and incubated for 24 hours. Uptake of each compound was substantial as measured by its decrease in the medium. The levels of free tryptophan, phenylalanine and tyrosine were determined in the tissues after the 24 hours incubation. Shikimate did not change the aromatic animo acid levels in carrot tissue, but did increase all three in tobacco (3-fold or more), indicating a less stringent feedback control in tobacco. Anthranilate and indole increased the tissue tryptophan levels in both species by at least 17-fold, showing that the flow from anthranilate and indole to tryptophan was apparently unhindered by enzymatic control mechanisms. When tryptophan levels were elevated in both carrot and tobaccotissues by anthranilate, indole or tryptophan addition, there was also an increase in free phyenylalanine and tyrosine. This might be due to the reversal of phenylalanine and tyrosine feedback inhibition of chorismate mutase by the high tryptophan in the tissue. Chorismate mutase activity in tobacco crude extracts could be inhibited by 66–90% by 1 mM phenylalanine and /or tyrosine. Tryptophan at 1 mM stimulated the enzyme activity by 1/3 and completely reversed the phenylalanine and/or tyrosine inhibition of enzyme activity. Chorsimate mutase activity amino acids under a variety of conditions. Phenylpyruvate increased the phenylalanine levels and p-hydroxyphenylpyruvate increased the tyrosine levels in carrot and tobacco tissues indicating that there was no feedback control of the last step in phenylalanine and tyrosine biosynthesis.     

INCORPORATION OF PHENYLALANINE, TYROSINE AND TRYPTOPHAN INTO PROTEIN OF HOMOGENATES FROM DEVELOPING RAT BRAIN: KINETICS OF INCORPORATION AND RECIPROCAL INHIBITION

The kinetics of the incorporation into protein of [³H]phenylalanine, [³H]tyrosine and [³H]tryptophan were studied with homogenates prepared from whole brain of 1-, 7-, 21- and 60-day-old rats. The maximal velocities (V_max)of incorporation of phenylalanine and tyrosine decreased and the apparent Michaelis-constants (K_m) for all three amino acids increased with increasing age of the rats. Tyrosine had the smallest and tryptophan the largest K_m values in all age groups. Phenylalanine competitively inhibited the incorporation of tyrosine, but tyrosine inhibited non-competitively the incorporation of phenylalanine. Tryptophan inhibited competitively the incorporation of phenylalanine, but at least partially non-competitively the incorporation of tyrosine. Phenylalanine and tyrosine did not significantly affect the incorporation of tryptophan in homogenates from 60-day-old rats. In 1-day-old rats only a very large excess of phenylalanine or tyrosine inhibited detectably. The K_i for phenylalanine in the incorporation of tyrosine was significantly smaller in 1- than in 60-day-old rats. In every case the inhibition presumably occurred at a single rate-limiting step in the complicated process of incorporation of amino acids into protein.     

Metabolism of Aromatic Amino Acids in Microorganisms

It was found that when Rhodotorula rubra IFO 0911 was grown in a phenylalanine medium, benzoic acid and p-hydroxybenzoic acid besides cinnamic acid were formed in the cultured both. The conversions of cinnamic acid into benzoic acid and of benzoic acid into p-hydroxybenzoic acid, and the degradation of p-hydroxybenzoic acid were demonstrated in intact cells of Rhodotorula rubra. These activities were observed in the cells grown on various media, including the medium containing no phenylalanine, and were found to be distributed widely in Rhodotorula. The cells of Rhodotorula rubra were also able to degrade p-coumaric acid, 3,4-dihydroxybenzoic acid (protocatechuic acid), p-hydroxyphenyl-acetic acid, 3-methoxy-4-hydroxycinnamic acid (ferulic acid) and 3-methoxy-4-hydroxybenzoic acid (vanillic acid). From these results, the metabolic pathways for phenylalanine and tyrosine in Rhodotorula were discussed.     

Dietary total aromatic amino acid requirement and tyrosine replacement value for phenylalanine in Indian major carp: Cirrhinus mrigala (Hamilton) fingerlings

Two 8‐week growth trials were conducted to determine total aromatic amino acid requirement and tyrosine replacement value for phenylalanine in Cirrhinus mrigala fingerlings. To determine the phenylalanine requirement, 20 fish were randomly stocked in triplicate groups in 55‐L indoor polyvinyl flow‐through circular tanks and fed six experimental diets containing graded levels of phenylalanine (5.0, 7.5, 10.0, 12.5, 15.0 and 17.5 g kg^?1, dry diet) with 10 g kg^?1 tyrosine. Maximum weight gain (287%), best FCR (1.44) and PER (1.74) occurred at 12.5 g kg^?1 dietary phenylalanine. Quadratic regression analysis of weight gain, FCR and PER data indicated phenylalanine requirement at 13.5, 12.9 and 12.7 g kg^?1 of dry diet, respectively. Protein deposition was significantly (P < 0.05) higher at 12.5 g kg^?1 dietary phenylalanine. Based on the above results, phenylalanine requirement of C. mrigala is recommended at 13.0 g kg^?1 of dry diet, corresponding to 32.5 g kg^?1 of protein. On the basis of the above requirement, a second experiment with a similar design was conducted using six diets containing graded levels of tyrosine (2.1, 4.0, 6.0, 8.0, 10.0 and 12.0 g kg^?1) with 13.0 g kg^?1 phenylalanine fixed in all diets to determine the phenylalanine replacement value with that of tyrosine. Maximum weight gain (315%), best FCR (1.47) and PER (1.69) was at 8.0 g kg^?1 dietary tyrosine. Quadratic regression analysis of weight gain, FCR and PER data indicated tyrosine requirement at 9.0, 8.4 and 8.2 g kg^?1 of dry diet, respectively. Protein deposition was significantly (P < 0.05) higher at 8.0 g kg^?1 dietary tyrosine. On the basis of the above results, 8.5 g kg^?1 tyrosine, corresponding to 21.3 g kg^?1 of protein, is taken as the optimum requirement and the replacement value is 39.53% on a weight and 36% on a molar basis. Thus, the total aromatic amino acid requirement is 21.5 g kg^?1 of diet, corresponding to 53.8 g kg^?1 of protein for optimum C. mrigala growth.     

Identification,characterization and functional expression of a tyrosine ammonia-lyase and its mutants from the photosynthetic bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

A tyrosine ammonia-lyase (TAL) enzyme from the photosynthetic bacterium Rhodobacter sphaeroides (RsTAL) was identified, cloned and functionally expressed in Escherichia coli, where conversion of tyrosine to p-hydroxycinnamic acid (pHCA) was demonstrated. The RsTAL enzyme is implicated in production of pHCA, which serves as the cofactor for synthesis of the photoactive yellow protein (PYP) in photosynthetic bacteria. The wild type RsTAL enzyme, while accepting both tyrosine and phenylalanine as substrate, prefers tyrosine, but a serendipitous RsTAL mutant identified during PCR amplification of the RsTAL gene, demonstrates much higher preference for phenylalanine as substrate and deaminates it to produces cinnamic acid. Sequence analysis showed the presence of three mutations: Met4 → Ile, Ile325 → Val and Val409 → Met in this mutant. Sequence comparison with Rhodobacter capsulatus TAL (RcTAL) shows that Val409 is conserved between RcTAL and RsTAL. Two single mutants of RsTAL, Val409 → Met and Val 409 → Ile, generated by site-directed mutagenesis, demonstrate greater preference for phenylalanine compared to the wild type enzyme. Our studies illustrate that relatively minor changes in the primary structure of an ammonia-lyase enzyme can significantly affect its substrate specificity.     

Role of the second coordination sphere residue tyrosine 179 in substrate affinity and catalytic activity of phenylalanine hydroxylase

Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing molecular oxygen and tetrahydropterin as a cofactor, and belongs to the aromatic amino acid hydroxylases family. The catalytic domains of these enzymes are structurally similar. According to recent crystallographic studies, residue Tyr179 in Chromobacterium violaceum phenylalanine hydroxylase is located in the active site and its hydroxyl oxygen is 5.1 Å from the iron, where it has been suggested to play a role in positioning the pterin cofactor. To determine the catalytic role of this residue, the point mutants Y179F and Y179A of phenylalanine hydroxylase were prepared and characterized. Both mutants displayed comparable stability and metal binding to the native enzyme, as determined by their melting temperatures in the presence and absence of iron. The catalytic activity (k_cat) of the Y179F and Y179A proteins was lower than wild-type phenylalanine hydroxylase by an order of magnitude, suggesting that the hydroxyl group of Tyr179 plays a role in the rate-determining step in catalysis. The K_M values for different tetrahydropterin cofactors and phenylalanine were decreased by a factor of 3–4 in the Y179F mutant. However, the K_M values for different pterin cofactors were slightly higher in the Y179A mutant than those measured for the wild-type enzyme, and, more significantly, the K_M value for phenylalanine was increased by 10-fold in the Y179A mutant. By the criterion of k_cat/K_Phe, the Y179F and Y179A mutants display 10% and 1%, respectively, of the activity of wild-type phenylalanine hydroxylase. These results are consistent with Tyr179 having a pronounced role in binding phenylalanine but a secondary effect in the formation of the hydroxylating species. In conjunction with recent crystallographic analyses of a ternary complex of phenylalanine hydroxylase, the reported findings establish that Tyr179 is essential in maintaining the catalytic integrity and phenylalanine binding of the enzyme via indirect interactions with the substrate, phenylalanine. A model that accounts for the role of Tyr179 in binding phenylalanine is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations AAAHs aromatic amino acid hydroxylases - BH₂ 7,8-dihydro-l-biopterin - BH₄ (6R)-5,6,7,8-tetrahydro-l-biopterin - CD circular dichroism - cPAH Chromobacterium violaceum phenylalanine hydroxylase - DMPH₄ 6,7-dimethyl-5,6,7,8-tetrahydropterin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - ES-MS electrospray ionization mass spectrometry - hPAH human phenylalanine hydroxylase - ICP-AE inductively coupled plasma atomic emission - 6-MPH₄ 6-methyl-5,6,7,8-tetrahydropterin - PAH phenylalanine hydroxylase - PH₄ tetrahydropterin - PKU phenylketonuria - RDS rate-determining step - TH tyrosine hydroxylase - THA 3-(2-thienyl)-l-alanine - TPH tryptophan hydroxylase - wt wild-type