Enzymatic cycling assay for phenylpyruvate

Enzymatic cycling assays for the determination of L-phenylalanine and phenylpyruvate in deproteinized tissue extracts are described. Assay 1 couples glutamine transaminase K with L-phenylalanine dehydrogenase. Assay 2 combines phenylalanine dehydrogenase, L-amino acid oxidase, and catalase. In both assays, tyrosine and some other amino acids (or their alpha-keto acid analogs) can replace phenylalanine (or phenylpyruvate) to a small extent. Thus, if phenylalanine is to be measured a correction must be made for the nonspecificity of the reaction. By removing phenylalanine on a cation-exchange column it was possible to measure phenylpyruvate in tissue extracts. Concentrations of phenylpyruvate (mumol/kg) in normal rat liver, kidney, and brain were 2.1 +/- 1.1 (n = 8), 1.8 +/- 0.4 (n = 4), and 3.3 +/- 0.6 (n = 4), respectively.     

Production of L-phenylalanine from phenylpyruvate using resting cells of Escherichia coli

Summary We describe the production of L-phenylalanine from phenylpyruvate using resting cells of a genetically modified strain of Escherichia coli. Fermentations were carried out by continuously raising the feeding rate of D-glucose. We reached a biomass of 10 g/l dry weight and an aminotransferase activity of 14000 U/l The maximum phenylalanine concentration achieved was 173 mmol/l with a phenylpyruvate molar conversion yield of 95%.Dedicated to Prof. Dr. Heinz Harnisch on the occasion of his 60th birthday     

Enzymatic determination of L-phenylalanine and phenylpyruvate with L-phenylalanine dehydrogenase

An enzymatic method is described for the determination of L-phenylalanine or phenylpyruvate using L-phenylalanine dehydrogenase. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine or the reductive amination of the 2-oxoacid, respectively. The stoichiometric coupling of the coenzyme allows a direct spectrophotometric assay of the substrate concentration. The equilibrium of the reaction favors L-phenylalanine formation; however, by measuring initial reaction velocities, the enzyme can be used for L-phenylalanine determination, too. Standard solutions of L-phenylalanine in the range of 10-300 microM and of phenylpyruvate (5-100 microM) show a linearity between the value for dENADH/min and the substrate concentration. Besides phenylalanine, the enzyme can convert tyrosine and methionine, and their oxoacids, respectively. The Km values of these substrates are higher. The influence of tyrosine on the determination of phenylalanine was studied and appeared tolerable for certain applications.     

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.     

The biochemical basis for growth inhibition by L-phenylalanine in Neisseria gonorrhoeae

Clinical isolates of Neisseria gonorrhoeae are commonly subject to growth inhibition by phenylpyruvate or by L-phenylalanine. A blockade of tyrosine biosynthesis is indicated since inhibition is reversed by either L-tyrosine or 4-hydroxyphenylpyruvate. Phenylalanine-resistant (PheR) and phenylalanine-sensitive (PheS) isolates both have a single 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase that is partially inhibited by L-phenylalanine (80%). However, PheS and PheR isolates differ in that the ratio of phenylpyruvate aminotransferase to 4-hydroxyphenylpyruvate aminotransferase is distinctly greater in PheS isolates than in PheR isolates. A mechanism for growth inhibition is proposed in which phenylalanine exerts two interactive effects. (i) Phenylalanine decreases precursor flow to 4-hydroxyphenylpyruvate through its controlling effect upon DAHP synthase; and (ii) phenylalanine is largely transaminated to phenylpyruvate, which saturates both aminotransferases, preventing transamination of an already limited supply of 4-hydroxyphenylpyruvate to L-tyrosine.     

Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase

A simple, rapid, accurate, and precise colorimetric assay for the determination of L-phenylalanine in plasma samples using L-phenylalanine dehydrogenase [L-phenylalanine:NAD+-oxidoreductase (deaminating)] from Rhodococcus sp. M 4 is described. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine. However, the equilibrium of reaction favors L-phenylalanine formation. By stoichiometric coupling of this reaction with diaphorase/iodonitro tetrazolium chloride (INT) the formed NADH converts INT to a formazan whereby the reaction is displaced in favor of phenylpyruvate. Using a kinetic approach the increase in absorbance at 492 nm shows linearity over more than 30 min. Deproteinized standard solutions of L-phenylalanine in the range from 30 to 1200 mumol/liter show a linearity between the dAformazan/30 min and the substrate concentration. In phenylketonuria (PKU) plasma samples no interferences caused by L-tyrosine or phenylpyruvic acid are seen. Applicability is demonstrated by comparative determination of plasma L-phenylalanine of treated PKU patients by the colorimetric method and automated amino acid analysis.     

Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes.

Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes. We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield. The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration. The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70 degrees C for at least 60 min or in the range of pH 5 to 10.8 at 50 degrees C for 10 min. The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism. The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate.     

A monofunctional and thermostable prephenate dehydratase from the archaeon Methanocaldococcus jannaschii

Prephenate dehydratase (PDT) is an important but poorly characterized enzyme that is involved in the production of L-phenylalanine. Multiple-sequence alignments and a phylogenetic tree suggest that the PDT family has a common structural fold. On the basis of its sequence, the PDT from the extreme thermophile Methanocaldococcus jannaschii (MjPDT) was chosen as a promising representative of this family for pursuing structural and functional studies. The corresponding pheA gene was cloned and expressed in Escherichia coli. It encodes a monofunctional and thermostable enzyme with an N-terminal catalytic domain and a C-terminal regulatory ACT domain. Biophysical characterization suggests a dimeric (62 kDa) protein with mixed alpha/beta secondary structure elements. MjPDT unfolds in a two-state manner (Tm = 94 degrees C), and its free energy of unfolding [DeltaGU(H2O)] is 32.0 kcal/mol. The purified enzyme catalyzes the conversion of prephenate to phenylpyruvate according to Michaelis-Menten kinetics (kcat = 12.3 s-1 and Km = 22 microM at 30 degrees C), and its activity is pH-independent over the range of pH 5-10. It is feedback-inhibited by L-phenylalanine (Ki = 0.5 microM), but not by L-tyrosine or L-tryptophan. Comparison of its activation parameters (DeltaH(++)= 15 kcal/mol and DeltaS(++)= -3 cal mol-1 K-1) with those for the spontaneous reaction (DeltaH(++) = 17 kcal/mol and DeltaS(++)= -28 cal mol-1 K-1) suggests that MjPDT functions largely as an entropy trap. By providing a highly preorganized microenvironment for the dehydration-decarboxylation sequence, the enzyme may avoid the extensive solvent reorganization that accompanies formation of the carbocationic intermediate in the uncatalyzed reaction.     

Metabolism of DL-(+/-)-phenylalanine by Aspergillus niger.

A fungus capable of degrading DL-phenylalanine was isolated from the soil and identified as Aspergillus niger. It was found to metabolize DL-phenylalanine by a new pathway involving 4-hydroxymandelic acid. D-Amino acid oxidase and L-phenylalanine: 2-oxoglutaric acid aminotransferase initiated the degradation of D- and L-phenylalanine, respectively. Both phenylpyruvate oxidase and phenylpyruvate decarboxylase activities could be demonstrated in the cell-free system. Phenylacetate hydroxylase, which required reduced nicotinamide adenine dinucleotide phosphate, converted phenylacetic acid to 2- and 4-hydroxyphenylacetic acid. Although 4-hydroxyphenylacetate was converted to 4-hydroxymandelate, 2-hydroxyphenylacetate was not utilized until the onset of sporulation. During sporulation, it was converted rapidly into homogentisate and oxidized to ring-cleaved products. 4-Hydroxymandelate was degraded to protocatechuate via 4-hydroxybenzoylformate, 4-hydroxybenzaldehyde, and 4-hydroxybenzoate.     

Biosynthesis of beta-phenethyl alcohol in Candida guilliermondii.

$\text{Candida guilliermondii}$ produced β-phenethyl alcohol and β-phenyllactic acid when grown in a synthetic medium containing L-phenylalanine as sole source of nitrogen. The cell-free preparations from these cells showed the following enzymes: phenylalanine aminotransferase, phenylpyruvate decarboxylase, phenylpyruvate reductase and phenylacetaldehyde reductase. The cell-free preparations of $\text{C. guilliermondii}$ grown in medium with ammonium sulfate, lacked these enzyme activities, indicating the inducible nature of these enzymes. The results indicate the role of β-phenylpyruvate as a key intermediate in the pathway of biosynthesis of β-phenethyl alcohol and β-phenyllactic acid from L-phenylalanine.     

Shifting the biotransformation pathways of L-phenylalanine into benzaldehyde by Trametes suaveolens CBS 334.85 using HP20 resin

AIMS: The biotransformation of L-phenylalanine into benzaldehyde (bitter almond aroma) was studied in the strain Trametes suaveolens CBS 334.85. METHODS AND RESULTS: Cultures of this fungus were carried out in the absence or in the presence of HP20 resin, a highly selective adsorbent for aromatic compounds. For the identification of the main catabolic pathways of L-phenylalanine, a control medium (without L-phenylalanine) was supplemented with each of the aromatic compounds, previously detected in the culture broth, as precursors. Trametes suaveolens CBS 334.85 was shown to biosynthesize benzyl and p-hydroxybenzyl derivatives, particularly benzaldehyde, and large amounts of 3-phenyl-1-propanol, benzyl and p-hydroxybenzyl alcohols as the products of both cinnamate and phenylpyruvate pathways. CONCLUSION: The addition of HP20 resin, made it possible to direct the catabolism of L- phenylalanine to benzaldehyde, the desired target compound, and to trap it before its transformation into benzyl alcohol. In these conditions, benzaldehyde production was increased 21-fold, from 33 to 710 mg l-1 corresponding to a molar yield of 31%. SIGNIFICANCE AND IMPACT OF THE STUDY: These results showed the good potential of Trametes suaveolens as a biotechnological agent to synthesize natural benzaldehyde which is one of the most important aromatic aldehydes used in the flavour industry.     

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.     

Rhodococcus L-phenylalanine dehydrogenase: kinetics, mechanism, and structural basis for catalytic specificity

Phenylalanine dehydrogenase catalyzes the reversible, pyridine nucleotide-dependent oxidative deamination of L-phenylalanine to form phenylpyruvate and ammonia. We have characterized the steady-state kinetic behavior of the enzyme from Rhodococcus sp. M4 and determined the X-ray crystal structures of the recombinant enzyme in the complexes, E.NADH.L-phenylalanine and E.NAD(+). L-3-phenyllactate, to 1.25 and 1.4 A resolution, respectively. Initial velocity, product inhibition, and dead-end inhibition studies indicate the kinetic mechanism is ordered, with NAD(+) binding prior to phenylalanine and the products' being released in the order of ammonia, phenylpyruvate, and NADH. The enzyme shows no activity with NADPH or other 2'-phosphorylated pyridine nucleotides but has broad activity with NADH analogues. Our initial structural analyses of the E.NAD(+).phenylpyruvate and E.NAD(+). 3-phenylpropionate complexes established that Lys78 and Asp118 function as the catalytic residues in the active site [Vanhooke et al. (1999) Biochemistry 38, 2326-2339]. We have studied the ionization behavior of these residues in steady-state turnover and use these findings in conjunction with the structural data described both here and in our first report to modify our previously proposed mechanism for the enzymatic reaction. The structural characterizations also illuminate the mechanism of the redox specificity that precludes alpha-amino acid dehydrogenases from functioning as alpha-hydroxy acid dehydrogenases.     

苯丙氨酸的脱氨酶法测定

方法的原理是：由某些微生物所产生的苯丙氨酸脱氨酶能使苯丙氨酸分子氧化脱氨,生成相应的苯丙酮酸。在合适的反应体系中,苯丙酮酸与三氯化铁反应生成的化合物呈蓝绿色,这种颜色的深浅与苯丙氨酸含量成正比,制备的标准曲线有良好的线性。因而可借此法对测试样品中的苯丙氨酸含量     

A simple spectrophotometric assay for arogenate dehydratase

A simple spectrophotometric assay for arogenate dehydratase, the enzyme that catalyzes the formation of L-phenylalanine from L-arogenate, is presented. The method couples the arogenate dehydratase reaction with that of an aromatic aminotransferase partially purified from Acinetobacter calcoaceticus. In the presence of 2-ketoglutarate, phenylpyruvate formation is measured at 320 nm at basic pH. The method was compared with two other methods already in use in our laboratory for arogenate dehydratase. The new method is simple, quick, fairly sensitive, and especially suitable for the screening of a large number of samples.     

Phenylacetate and the enduring behavioral deficit in experimental phenylketonuria

Phenylacetate, a metabolite derived from phenylalanine, is clearly associated with brain dysfunction in simulated phenylketonuria. Injections of phenylacetate, phenylethylamine, or p-chlorophenylalanine + L-phenylalanine, all yielding similar concentrations of phenylacetate in the rat brain during post-natal development, induced similar behavioral deficits: hypoactivity in an open field and poor performance in both a water maze and shuttle box. In contrast, animals treated with the other major metabolites of phenylalanine, phenylpyruvate, phenyllactate and mandelate, during the same developmental period displayed normal behavior.     

Exploitation of the broad specificity of the membrane-bound isoenzyme of lactate dehydrogenase for direct selection of null mutants in Neisseria gonorrhoeae

Lactic acid is readily utilized as a carbon and energy source by Neisseria gonorrhoeae. The oxidation of lactate is coupled to electron transport via a membrane-bound lactate dehydrogenase (iLDH) which is independent of pyridine nucleotide. The broad substrate specificity of iLDH endows N. gonorrhoeae with the novel ability to convert phenyllactate to L-phenylalanine via phenylpyruvate. N. gonorrhoeae ATCC 27628 typifies a class of clinical isolate whose growth is inhibited by phenylpyruvate (or L-phenylalanine). Exploiting resistance to growth inhibition by phenyllactate as a strategy of positive selection, mutant derivatives of strain ATCC 27628 lacking iLDH activity were readily obtained. These mutants are incapable of oxidizing phenyllactate, and lack the parent-strain ability to reduce c-type cytochromes in the presence of lactate, phenyllactate or 4-hydroxyphenyllactate. They retain, however, a cytoplasmic NAD(+)-linked lactate dehydrogenase (nLDH). Since the mutants retained the ability to grow on lactate as a sole source of carbon, nLDH presumably can function in an opposite-to-normal physiological direction in the absence of iLDH. This would explain the failure to isolate iLDH-deficient mutants by selection for inability to grow on lactate.     

A multispecific quintet of aromatic aminotransferases that overlap different biochemical pathways in Pseudomonas aeruginosa

Pseudomonas aeruginosa possesses dual enzymatic sequences to both L-phenylalanine and L-tyrosine, a biosynthetic arrangement further complicated by the presence of five aromatic aminotransferases. Each aminotransferase is capable of transamination in vitro with any of the three keto acid intermediates in the aromatic pathway (phenylpyruvate, 4-hydroxyphenylpyruvate, or prephenate). The fractional contribution of these aminotransferases to particular transamination reactions in vivo can best be approached through the systematic and sequential elimination of individual aminotransferase activities by mutation. A program of sequential mutagenesis has produced two aminotransferase-deficient mutations. The first mutation imposed a phenotype of bradytrophy for L-phenylalanine (doubling time of 2.4 h in minimal salts/glucose medium compared to a 1.0-h doubling time for wild type). This mutant completely lacked an enzyme denoted aminotransferase AT-2. A genetic background of aminotransferase AT-2 deficiency was used to select for a second mutation which produced a phenotype of multiple auxotrophy for L-phenylalanine, L-aspartate, and L-glutamate. The double mutant completely lacked activity for aromatic aminotransferase AT-1 in addition to the missing aminotransferase AT-2. Enzymes AT-1 (Mr = 64,000) and AT-2 (Mr = 50,000) were readily separated from one another by gel filtration and were individually characterized for pH optima, freeze-thaw stability, heat lability, and molecular weight. The phenotypic and enzymological characterizations of the aminotransferase mutants strongly support the primary in vivo role of enzyme AT-2 in L-phenylalanine and L-tyrosine biosynthesis, while enzyme AT-1 must primarily be engaged in L-aspartate and L-glutamate synthesis. The substrate specificities and possible in vivo functions for AT-3, AT-4, and AT-5 are also considered.     

Aromatic hydroxylation of phenylalanine as an assay for hydroxyl radicals: application to activated human neutrophils and to the heme protein leghemoglobin

Attack of hydroxyl radical (.OH), generated by a Fenton system at physiological pH, upon L-phenylalanine produces three isomeric tyrosines, o-tyrosine (2-hydroxyphenylalanine), m-tyrosine (3-hydroxyphenylalanine), and p-tyrosine (4-hydroxyphenylalanine). These may be separated by high-performance liquid chromatography and measured using an electrochemical detector. Since L-phenylalanine is relatively nontoxic, it is proposed that generation of these three tyrosines from phenylalanine can be used as an assay for .OH in biological systems. The use of the assay to measure .OH production by leghemoglobin (plus H2O2) and by activated human neutrophils is described. No .OH production by activated human neutrophils was observed unless a source of iron ions was added to the reaction mixture, which suggests that these cells do not release an iron "promoter" of .OH generation from superoxide and hydrogen peroxide.     

Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli

With L-aspartate (L-Asp) as the amino donor, L-phenylalanine (L-Phe) can be prepared from phenylpyruvate (PPA) via an amination reaction mediated by aminotransferase (encoded by aspC). On the other hand, L-Asp can be produced by an aspartase (encoded by aspA) -catalyzed reaction using fumaric acid as substrate. To overproduce aspartase in Escherichia coli, the aspA gene was cloned and overexpressed 180 times over the wild-type level. The use of AspA-overproducing E. coli strain for L-Asp production exhibited an 83% conversion, approaching to the theoretical yield, whereas the wild-type strain obtained scarcely L-Asp. Furthermore, the recombinant strain overproducing both AspA and AspC was able to produce L-Asp and L-Phe simultaneously by using fumaric acid and PPA as substrates. As a result, the conversion yields obtained for L-Asp and L-Phe were 78% and 85%, respectively. In sharp contrast, the wild-type strain attained a conversion of L-Phe less than 15% and an undetectable level of L-Asp. This result illustrates a potential and attractive process to yield both L-Asp and L-Phe by coupling AspA and AspC. A further study on the repeated use of the recombinant strain immobilized with calcium alginate showed that after eight batch runs L-Asp conversion maintained roughly constant (around 75%), whereas L-Phe conversion dropped to 65% from 81%. This result indicates the stability of AspA being superior to AspC.