Selective production of phenylalanine from phenylpyruvate using growing cells ofCorynebacterium glutamicum

Summary Phenylalanine was produced from phenylpyruvate by growing cells of a mutant strain ofCorynebacterium glutamicum over-producing valine. A defined medium was used to minimize the accumulation of valine. The maximum phenylalanine concentration achieved was 44.5 mM (7.5 g/l) with a phenylpyruvate molar conversion of 75%.     

Effect of phenylalanine and its metabolites on ATP diphosphohydrolase activity in synaptosomes from rat cerebral cortex

The in vitro effects of phenylalanine and some of its metabolites on ATP diphosphohydrolase (apyrase, EC 3.6.1.5) activity in synaptosomes from rat cerebral cortex were investigated. The enzyme activity in synaptosomes from rats subjected to experimental hyperphenylalaninemia (-methylphenylalanine plus phenylalanine) was also studied. In the in vitro studies, a biphasic effect of phenylalanine on both enzyme substrates (ATP and ADP) was observed, with maximal inhibition at 2.0 mM and maximal activation at 5.0 mM. Inhibition of the enzyme activity was not due to calcium chelation. Moreover, phenylpyruvate, when compared with phenylalanine showed opposite effects on the enzyme activity, suggesting that phenylalanine and phenylpyruvate bind to two different sites on the enzyme. The other tested phenylalanine metabolites (phenyllactate, phenylacetate and phenylethylamine) had no effect on ATP diphosphohydrolase activity. In addition, we found that ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats with chemically induced hyperphenylalaninemia was significantly enhanced by acute or chronic treatment. Since it is conceivable that ATPase-ADPase activities play an important role in neurotransmitter (ATP) metabolism, it is tempting to speculate that our results on the deleterious effects of phenylalanine and phenylpyruvate on ATP diphosphohydrolase activity may be related to the neurological dysfunction characteristics of naturally and chemically induced hyperphenylalaninemia.     

Parallel control of hepatic proteolysis by phenylalanine and phenylpyruvate through independent inhibitory sites at the plasma membrane.

Intracellular protein degradation in the rat hepatocyte is regulated by 7 amino acids of which Leu, Gln, and Tyr play major roles. Although Phe is known to inhibit proteolysis as effectively as Tyr at high concentrations, it has not been regarded as an active regulator because of its rapid hydroxylation to Tyr. We now show that proteolytic responses to Phe during liver perfusion differ strikingly from effects of the multiphasic regulators Leu, Gln, and Tyr in eliciting mirror image responses at half-normal and normal plasma concentrations. Since response curves to phenylpyruvate and Phe were identical, we considered the possibility that phenylpyruvate mediated its anomalous inhibition intracellularly. However, when phenylpyruvate was produced from phenyllactate intracellularly at a rate providing the same rate of transamination (and intracellular concentration) as that derived from the uptake of phenylpyruvate, no response was obtained. Hence, the effect of phenylpyruvate was not initiated within the cell but more likely from the plasma membrane. Comparable evidence for Phe is less direct. Recent findings indicate that recognition sites for Leu and Gln are located at the plasma membrane. Since Phe augments the concerted inhibition by Leu and Gln at 4-fold normal levels, Phe is probably recognized in close proximity to them. However, the failure of phenylpyruvate to substitute for Phe in this interaction suggests that proteolytic inhibition by phenylpyruvate and Phe is mediated through similar, although independent, plasma membrane sites.     

Phenylalanine and tyrosine metabolism in the facultative methylotroph Nocardia sp. 239

Nocardia sp. 239 is able to use l-tyrosine and both d- and l-phenylalanine as carbon-, energy- and nitrogen sources for growth. The catabolism of these compounds is by way of (4-hydroxy)phenylpyruvate and (4-hydroxy)-phenylacetate as intermediates and the pathways merge at the level of homogentisate. The conversion of the amino acids into (4-hydroxy)phenylpyruvate is catalyzed by an inducible NAD-dependent phenylalanine dehydrogenase and l-tyrosine aminotransferase, respectively. Incubation of the organism in media with l-phenylalanine plus phenyl-pyruvate resulted in diauxic growth, with phenylpyruvate used first. Phenylalanine dehydrogenase activity cold only be detected after depletion of phenylpyruvate, in the ensuing second growth phase on l-phenylalanine. During growth on phenylalanine plus methanol, low levels of phenylalanine dehydrogenase were detected and this resulted in simultaneous utilization of the two substrates. Following diepoxyoctane treatment, mutants of Nocardia sp. 239 affected in phenylalanine and phenylpyruvate degradation were isolated. Double mutants blocked in both phenylalanine dehydrogenase and phenylpyruvate decarboxylase completely failed to catabolize phenylalanine. The absence of these enzymes did not affect growth on tyrosine.Abbreviations RuMP ribulose monophosphate - EMS ethylmethanesulphonate - NTG N-methyl-N-nitro-N-nitrosoguanidine     

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.     

Identification of a novel glyoxylate reductase supports phylogeny-based enzymatic substrate specificity prediction

Phylogenetic analysis of the superfamily of D-2-hydroxyacid dehydrogenases identified the previously unrecognized cluster of glyoxylate/hydroxypyruvate reductases (GHPR). Based on the genome sequence of Rhizobium etli, the nodulating endosymbiont of the common bean plant, we predicted a putative 3-phosphoglycerate dehydrogenase to exhibit GHPR activity instead. The protein was overexpressed and purified. The enzyme is homodimeric under native conditions and is indeed capable of reducing both glyoxylate and hydroxypyruvate. Other substrates are phenylpyruvate and ketobutyrate. The highest activity was observed with glyoxylate and phenylpyruvate, both having approximately the same kcat/Km ratio. This kind of substrate specificity has not been reported previously for a GHPR. The optimal pH for the reduction of phenylpyruvate to phenyllactate is pH 7. These data lend support to the idea of predicting enzymatic substrate specificity based on phylogenetic clustering.     

PYRUVATE METABOLISM BY HOMOGENATES OF HUMAN BRAIN: EFFECTS OF PHENYLPYRUVATE AND IMPLICATIONS FOR THE ETIOLOGY OF THE MENTAL RETARDATION IN PHENYLKETONURIA

Abstract— The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by homogenates of human brain was investigated. In the presence of 5 mM pyruvate as substrate homogenates of human cerebral cortex fixed about 1 μmol of H¹⁴CO₃^-_- per g of tissue in 30 min. Phenylpyruvate at a concentration of 5 raw inhibited the fixation of H¹⁴ CO₃^-_- by homogenates of human brain by approximately 50 per cent, whereas 5 mM phenylalanine had no effect. The inhibition of pyruvate carboxylation by phenylpyruvate was dependent upon the concentration of the inhibitor. The activity of pyruvate carboxylase (EC 6.4.1.1) in human cerebral cortex was 02–0.4 units, with a K_m for pyruvate of about 0.2 mM. Homogenates of human cerebral cortex decarboxylated [1-¹⁴C]pyruvate to ¹⁴CO₂ at a rate of about 5 μmol per g of tissue per 15 min, with a 20–50 per cent reduction in the presence of 5 mM phenylpyruvate; phenylalanine at the same concentration had no effect. The possible toxic effect of phenylpyruvate on the metabolism of pyruvate in the brains of untreated phenylketonuric patients is discussed.     

Differences in properties between aromatic amino acid: Aromatic keto acid aminotransferases and aromatic amino acid: α-ketoglutarate aminotransferases

Homogenates of rat liver transaminate phenylpyruvate (PP), as well as α-ketoglutarate (α-KG), in the presence of L-tyrosine, 3,4-dihydroxyphenylalanine (L-DOPA) or L-tryptophan. Aminotransferase activity with phenylpyruvate and DOPA, but not with tyrosine, was inhibited by excess phenylpyruvate. Tyrosine and DOPA aminotransferase activities with phenylpyruvate were more heat stable than the corresponding activities with α-ketoglutarate. Aminotransferase activities with phenylpyruvate were not significantly induced following intraperitoneal injections of cortisol, glucagon or serotonin, compared with a 3 to 7-fold increase in the aminotransferase activities with α-ketoglutarate. Tyrosine:phenylpyruvate aminotransferase activity rose 40% at night, compared with a 300% increase in tyrosine:α-ketoglutarate aminotransferase activity. The results suggest that aminotransferases catalysing transfers between aromatic keto acids and aromatic amino acids are separate enzymes from those utilizing α-ketoglutarate as the acceptor keto acid.     

Ketone bodies affect the enzymatic activity of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF), a long known proinflammatory cytokine exhibits perplexing enzymatic activities: tautomeric conversion of D-dopachrome and phenylpyruvate. Whether these catalytic activities bear functional relevance regarding MIF's multifaceted roles is under current scrutiny. Nevertheless, intense search has already started for pharmacological agents that target MIF's tautomerase activity. We have probed several antiinflammatory compounds against keto--enol (enolase) and enol--keto (ketonase) conversion of phenylpyruvate by MIF with spectrophotometry. We have identified acidic CH groups as markers of inhibitor potency toward MIF phenylpyruvate tautomerase. Among simple model molecules with strong acidic CH groups we found acetylacetone the best inhibitor particularly against the ketonase activity. Ketones of physiological importance - ketone bodies - also feature acidic CH groups and have been reported to exert certain anti-inflammatory effects. In this paper we report that ketone bodies inhibit preferentially the ketonase activity of MIF in vitro. Future studies should address whether such an interaction might operate in vivo and delineate its possible relevance concerning cytokine and non-cytokine roles of MIF.     

Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of (14)CO(2) from [1-(14)C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K(i) of 100mum. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

THE EFFECTS OF PHENYLKETONURIC AND OTHER METABOLITES ON SULFATED GALACTOCEREBROSIDE SYNTHESIS IN VIVO AND IN CULTURE

Abstract— Sulfated galactocerebroside synthesis was examined in vitro in mouse spinal cord cultures. This system permitted the study of the effects of phenylketonuric metabolites upon synthesis of a specific myelin component, sulfatide, formed early in postnatal development in mice. A significant reduction of Na₂³⁵SO₄ incorporation into myelin sulfatide was observed when spinal cord cultures were grown in the presence of 1000 μm -l -phenylalanine and 500 μm -phenylpyruvate (51 and 700%, respectively). No reduction was observed with β-phenyllactate (300 μm and) phenylacetate (250 μm ). Light microscopy indicated that the phenylpyruvate and phenylalanine treated cultures were less extensively myelinated compared to control and β-phenyllactate or phenylacetate treated cultures. The reduction of sulfatide synthesis by phenylpyruvate was shown to be reversible. Intracerebral bilateral injections (8 μg) of l -phenylalanine, phenylpyruvate, α-ketobutyrate, α-ketoisocaproate, α-ketoisovalerate, β-phenyllactate, and phenylacetate in mice 8–15 days old, followed by i.p. administration of radioactive sulfate, resulted in significantly reduced incorporation (all P < 0.05) of sulfate into brain sulfatides with all compounds tested with the exception of β-phenyllactate and phenylacetate. In adult mouse, phenylpyruvate treatment also resulted in a significant decrease in labelling of brain sulfatide. The effects of phenylpyruvate and other metabolites upon pyruvate oxidation in mouse brain homogenates were examined by measuring ¹⁴CO₂ release from [1-¹⁴C]pyruvate. Both phenylpyruvate and α-ketoisocaproate at 1 × 10^-3 resulted in a decrease in ¹⁴CO₂ produced, while phenylacetate and β-phenyllactate had no effect. Sulfate incorporation into sulfatide was reduced by α-ketoisocaproate and phenylpyruvate, and to a lesser extent by phenylalanine, α-ketobutyrate, and α-ketoisovalerate. Phenyllactate and phenylacetate had no effect, either in vivo, or in culture. This order of effectiveness may be related in part to the effects of these compounds on pyruvate oxidation.     

Functional significance of aromatic amino acid: aromatic keto acid aminotransferases in rat brain and liver: competition for tryptophan between aminotransferases and tryptophan hydroxylase in vitro.

Using low concentrations of substrates and cofactors, a comparison was made of the relative rates by which aminotransferases catalysed transaminations between aromatic amino acids and aromatic or aliphatic keto acids. Tryptophan aminotransferase in homogenates of rat midbrain and liver transaminated phenylpyruvate at a rate 70 to 150-fold greater than the rate with α-ketoglutarate at low concentrations of substrates. Phenylalanine aminotransferase in liver and midbrain also was more active with aromatic keto acids than with aliphatic keto acids. However, tyrosine aminotransferase in dialysed homogenates of midbrain transaminated α-ketoglutarate and phenylpyruvate at approximately equal rates. Fresh homogenates of midbrain contained an inhibitor which markedly decreased tyrosine aminotransferase activity with α-ketoglutarate but not with phenylpyruvate. Tyrosine aminotransferase in homogenates of rat liver transaminated α-ketoglutarate and phenylpyruvate at equal rates below 10 μM keto acid, but above 10 μM, transamination of α-ketoglutarate was favoured. With homogenates of liver, transamination of α-ketoglutarate, but not phenylpyruvate, by tyrosine was increased 650% by exogenous pyridoxal phosphate. Since tryptophan aminotransferase in the brain may compete with tryptophan hydroxylase for available tryptophan, a comparison was made of the relative activities of tryptophan hydroxylase and tryptophan aminotransferase. At concentrations above 7.5 μM phenylpyruvate, transamination was 8 to 17-fold greater than the rate of hydroxylation of 50 μM tryptophan.     

Impairment of hepatic pyruvate metabolism in phenylketonuria

In patients with untreated classical phenylketonuria, elevated plasma levels of pyruvate, lactate, phenylalanine and phenylpyruvate were observed. After about 10 days on a low-phenylalanine diet, the levels of pyruvate, lactate and phenylpyruvate in plasma of treated patients returned to normal; the concentrations of phenylalanine in plasma were markedly lowered. In plasma from hyperphenylalaninemic subjects, phenylpyruvate was not detectable; pyruvate and lactate were within normal limits. Phenylpyruvate at a concentration of about 1 mM inhibited pyruvate carboxylation by human and rat liver homogenates by about 50%; phenylalanine had no effect on this process. The values of apparent Km for pyruvate and Ki for phenylpyruvate of human liver pyruvate carboxylase were approximately 0.27 mM and 1.4 mM, respectively. These studies suggest an impairment in hepatic pyruvate metabolism in untreated phenylketonuric patients.     

Characterization of D-lactate dehydrogenase producing D-3-phenyllactic acid from Pediococcus pentosaceus

D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s(-1), and 100 (mmol/L)(-1) s(-1) respectively.     

Energy metabolism in the brains of L-phenylalanine-treated chicks

Abstract— When day-old chicks were injected intraperitoneally with 1.62mmoles of l -phenylalanine, they developed a condition resembling narcosis. Simultaneously, whole brain levels of phenylalanine were 2–4 μmol/g, whereas those in control brain were 0.06 μmol/g. Examination of some glycolytic intermediates in the brain revealed significant decreases in fructose-1,6-diphosphate, l -α-glycerol phosphate and lactate, in comparison to the levels of these compounds in the saline-injected control animals. Levels of glucose and glucose-6-phosphate either increased or did not change, whereas levels of glycogen did not differ significantly. Phosphocreatine increased reciprocally with the decrease in inorganic phosphate. The levels of adenine nucleotide (energy charge) were not affected. Utilization of cerebral high-energy phosphates was depressed by 50–70 per cent when determined as a function of metabolic rate in the brain at 15- and 30-s periods of ischaemia according to the ‘closed-system’ technique. Explanations for these data have been examined, such as toxicity of phenylacetate and inhibition of glycolytic enzymes by phenylpyruvate and l -phenylalanine and their relevance to this study is discussed.     

The effect of phenylpyruvate on pyruvate metabolism in rat brain

1. The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by isolated mitochondria from rat brain was investigated. 2. Phenylpyruvate inhibited the fixation of H(14)CO(3) (-) in the presence of pyruvate by intact rat brain mitochondria, whereas phenylalanine and other metabolites of this amino acid had no inhibitory effect on this process. 3. Pyruvate carboxylase activity in freeze-dried rat brain mitochondrial preparations was also inhibited only by phenylpyruvate, and a ;mixed type' inhibition was observed. 4. The K(m) for pyruvate of rat brain pyruvate carboxylase was about 0.2mm. 5. The concentration of phenylpyruvate required for a 50% inhibition of H(14)CO(3) (-) fixation by the intact mitochondria and of pyruvate carboxylase activity was dependent on the concentration of pyruvate used in the incubation medium. 6. The possible significance of inhibition of pyruvate carboxylase activity by phenylpyruvate in the brains of phenylketonuric patients is discussed.     

Production of l-phenylalanine from phenylpyruvate by Paracoccus denitrificans containing aminotransferase activity

Summary To establish an efficient production method for l-phenylalanine, the production of l-phenylalanine from phenylpyruvate by Paracoccus denitrificans pFPr-1 containing aminotransferase activity was investigated. By using intact cells, 0.74M l-phenylalanine was produced from 0.8M phenylpyruvate (conversion yield, 92.5%). Moreover, by using immobilized cells with -carrageenan, when the space velocity was 0.1 h^-1 at 30°C, 0.135 M l-phenylalanine was produced from 0.15 M phenylpyruvate (conversion yield, 90%). The half-life of the l-phenylalanine-forming activity of the column was estimated to be about 30 days at 30°C.     

Characterization of a thiamin diphosphate-dependent phenylpyruvate decarboxylase from Saccharomyces cerevisiae

The product of the ARO10 gene from Saccharomyces cerevisiae was initially identified as a thiamine diphosphate-dependent phenylpyruvate decarboxylase with a broad substrate specificity. It was suggested that the enzyme could be responsible for the catabolism of aromatic and branched-chain amino acids, as well as methionine. In the present study, we report the overexpression of the ARO10 gene product in Escherichia coli and the first detailed in vitro characterization of this enzyme. The enzyme is shown to be an efficient aromatic 2-keto acid decarboxylase, consistent with it playing a major in vivo role in phenylalanine, tryptophan and possibly also tyrosine catabolism. However, its substrate spectrum suggests that it is unlikely to play any significant role in the catabolism of the branched-chain amino acids or of methionine. A homology model was used to identify residues likely to be involved in substrate specificity. Site-directed mutagenesis on those residues confirmed previous studies indicating that mutation of single residues is unlikely to produce the immediate conversion of an aromatic into an aliphatic 2-keto acid decarboxylase. In addition, the enzyme was compared with the phenylpyruvate decarboxylase from Azospirillum brasilense and the indolepyruvate decarboxylase from Enterobacter cloacae. We show that the properties of the two phenylpyruvate decarboxylases are similar in some respects yet quite different in others, and that the properties of both are distinct from those of the indolepyruvate decarboxylase. Finally, we demonstrate that it is unlikely that replacement of a glutamic acid by leucine leads to discrimination between phenylpyruvate and indolepyruvate, although, in this case, it did lead to unexpected allosteric activation.