Production of l-Phenylalanine from trans-Cinnamic Acid with Rhodotorula glutinis Containing l-Phenylalanine Ammonia-Lyase Activity

An enzymatic method using l-phenylalanine ammonia-lyase (EC 4.3.1.5) for the rapid conversion of trans-cinnamic acid to l-phenylalanine has been investigated. With Rhodotorula glutinis, enzyme activity as high as 0.3 U/ml of culture broth was obtained. The enzyme activity was kept stable for a relatively long time during cultivation by the addition of l-isoleucine. Optimization of the parameters of the conversion reaction resulted in accumulation of 18 mg of l-phenylalanine per ml of reaction mixture. The conversion yield from trans-cinnamic acid was about 70%. The method may provide a rapid and practical way to produce l-phenylalanine useful as an essential amino acid.     

Phosphatidylinositol Phosphodiesterase (Phospholipase C) Activity in the Pineal Gland: Characterization and Photoneural Regulation

Phosphatidylinositol phosphodiesterase (PL-C) appears to be a key element in the adrenergic regulation of pineal cyclic AMP levels. In the present study, the rat pineal enzyme was characterized using exogenous [3H]phosphatidylinositol (0.5 mM) as substrate. Half the enzyme activity was found in the cytosolic fraction, but the highest specific concentration was associated with the membrane fraction. Two pH optima (5.5 and 7.5) of enzyme activity were observed for the membrane fraction but only one in the cytosol fraction (pH 5.5). Enzyme activity in both fractions was Ca2+ dependent. In the case of the membrane protein in pH 7.5, the enzyme activity was sensitive to changes in Ca2+ in the 10-100 nM range. Addition of an equimolar concentration of phosphatidylinositol 4-phosphate nearly completely inhibited the hydrolysis of [3H]phosphatidylinositol; other phospholipids (1.0 mM) were less potent. This may reflect our present finding that [3H]phosphatidylinositol 4-phosphate is a better substrate than [3H]phosphatidylinositol for the enzyme. Stimulus deprivation (2 weeks of constant light or superior cervical ganglionectomy) reduced the cytosolic activity by 30% and had no effect on the membrane-associated enzyme.     

Mis-regulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase does not account for growth inhibition by phenylalanine in Agmenellum quadruplicatum

The growth of the blue-green bacterium, Agmenellum quadruplicatum, is inhibited in the presence of l-phenylalanine. This species has a single, constitutively synthesized 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase. l-Phenylalanine inhibits DAHP synthetase non-competitively with respect to both substrate reactants. Other aromatic amino acids do not inhibit the activity of DAHP synthetase. A common expectation for branch-point enzymes such as DAHP synthetase is a balanced pattern of feedback control by all of the ultimate end products. It seemed likely that growth inhibition might equate with defective regulation within the branched aromatic pathway. Accordingly, the possibility was examined that mis-regulation of DAHP synthetase by l-phenylalanine in wild-type cells causes starvation for precursors of the other aromatic end products. However, the molecular basis for growth inhibition cannot be attributed to l-phenylalanine inhibition of DAHP synthetase for the following reasons: (i) DAHP synthetase enzymes from l-phenylalanine-resistant mutants are more, rather than less, sensitive to feedback inhibition by l-phenylalanine. (ii) Shikimate not only fails to antagonize inhibition, but is itself inhibitory. (iii) Neither the sensitivity nor the completeness of l-phenylalanine inhibition of the wild-type enzyme in vitro appears sufficient to account for the potent inhibition of growth in vivo by l-phenylalanine. The dominating effect of l-phenylalanine in the control of DAHP synthetase appears to reflect a mechanism that prevents rather than causes growth inhibition by l-phenylalanine. The alteration of the control of DAHP synthetase in mutants selected for resistance to growth inhibition by l-phenylalanine did indicate that the cause for this metabolite vulnerability can be localized within the aromatic amino acid pathway. Apparently, an aromatic intermediate (between shikimate and the end products) accumulates in the presence of l-phenylalanine, causing toxicity by some unknown mechanism. It is concluded that phenylpyruvate, potentially formed by transamination of l-phenylalanine, is an unlikely cause of growth inhibition. Although several significant questions remain unanswered, our results suggest that single-effector control of DAHP synthetase, the first regulatory enzyme activity of a branched pathway, may be more appropriate than it would seem a priori.     

Chick pineal melatonin synthesis: light and cyclic AMP control abundance of serotonin N-acetyltransferase protein

Melatonin production in the pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87], the penultimate enzyme in melatonin synthesis. The rhythm is generated by an endogenous circadian clock. In the chick, a clock is located in the pinealocyte, which also contains two phototransduction systems. One controls melatonin production by adjusting the clock and the other acts distal to the clock, via cyclic AMP mechanisms, to switch melatonin synthesis on and off. Unlike the clock in these cells, cyclic AMP does not appear to regulate activity by altering AA-NAT mRNA levels. The major changes in AA-NAT mRNA levels induced by the clock seemed likely (but not certain) to generate comparable changes in AA-NAT protein levels and AA-NAT activity. Cyclic AMP might also regulate AA-NAT activity via changes in protein levels, or it might act via other mechanisms, including posttranslational changes affecting activity. We measured AA-NAT protein levels and enzyme activity in cultured chick pineal cells and found that they correlated well under all conditions. They rose and fell spontaneously with a circadian rhythm. They also rose in response to agents that increase cyclic AMP. They were raised by agents that increase cyclic AMP, such as forskolin, and lowered by agents that decrease cyclic AMP, such as light and norepinephrine. Thus, both the clock and cyclic AMP can control AA-NAT activity by altering the total amount of AA-NAT protein. Effects of proteosomal proteolysis inhibitors suggest that changes in AA-NAT protein levels, in turn, reflect changes in the rate at which the protein is destroyed by proteosomal proteolysis. It is likely that cyclic AMP-induced changes in AA-NAT protein levels mediate rapid changes in chick pineal AA-NAT activity. Our results indicate that light can rapidly regulate the abundance of a specific protein (AA-NAT) within a photoreceptive cell.     

The role of l-phenylalanine in the production of isobutene by Rhodotorula minuta

The role of l-phenylalanine and its synergistic effect on the production of isobutene were investigated with both the living cells and a cell-free system of Rhodotorula minuta IFO 1102. Many aromatic carboxylic acids also had the same effect on the production of isobutene as l-phenylalanine. Cycloheximide, an inhibitor of protein synthesis, inhibited the synergistic effect of l-phenylalanine on the production of isobutene. Furthermore, the cell-free extract prepared from cells cultivated in the medium which contained l-phenylalanine had isobutene-forming activity. These results confirm that l-phenylalanine is an inducer of a tentative “isobutene-forming enzyme” in Rhodotorula minuta.     

Production of p-hydroxycinnamic acid from glucose in Saccharomyces cerevisiae and Escherichia coli by expression of heterologous genes from plants and fungi

Biological production of p-hydroxycinnamic acid (pHCA) from glucose can be achieved via deamination of the aromatic amino acids l-tyrosine or l-phenylalanine. Deamination of l-phenylalanine produces trans-cinnamic acid (CA) which is further hydroxylated in the para position to produce pHCA. However, when tyrosine is used as the substrate, trans-pHCA is produced in one step. This reaction is accomplished by phenylalanine ammonia-lyase (PAL)/tyrosine ammonia-lyase (TAL). Various bacteria and eukaryotic microorganisms were screened for their ability to produce a PAL/TAL enzyme with high TAL activity. Cell-free extracts of the yeast Rhodotorula glutinis possessed the highest level of TAL activity (0.0143U/mg protein) and the lowest PAL/TAL ratio (1.68) amongst species examined. The gene for this enzyme was cloned and expressed in Escherichia coli and the kinetics of the purified PAL/TAL determined. The recombinant PAL/TAL possessed characteristics similar to those of the wild-type enzyme. Functional expression of R. glutinis PAL/TAL enzyme in Saccharomyces cerevisiae cells containing the plant C4H P-450 and P-450 reductase enzymes from Helianthus tuberosus allowed conversion of glucose to pHCA. Addition of l-phenylalanine to these cultures increased pHCA production confirming its production via the PAL route. When R. glutinis PAL/TAL was synthesized in an E. colil-phenylalanine producing strain (ATCC 31882) and grown on glucose, pHCA was formed in the absence of the Cytochrome P-450 and the P-450 reductase enzymes underlining its production via the TAL route without CA intermediacy.     

Increased Cyclic AMP-Dependent Protein Kinase Activity in Postmortem Brain from Patients with Bipolar Affective Disorder

Previous observations of reduced [3H]cyclic AMP binding in postmortem brain regions from bipolar affective disorder subjects imply cyclic AMP-dependent protein kinase function may be altered in this illness. To test this hypothesis, basal and stimulated cyclic AMP-dependent protein kinase activity was determined in cytosolic and particulate fractions of postmortem brain from bipolar disorder patients and matched controls. Maximal enzyme activity was significantly higher (104%) in temporal cortex cytosolic fractions from bipolar disorder brain compared with matched controls. In temporal cortex particulate fractions and in the cytosolic and particulate fractions of other brain regions, smaller but statistically nonsignificant increments in maximal enzyme activity were detected. Basal cyclic AMP-dependent protein kinase activity was also significantly higher (40%) in temporal cortex cytosolic fractions of bipolar disorder brain compared with controls. Estimated EC50 values for cyclic AMP activation of this kinase were significantly lower (70 and 58%, respectively) in both cytosolic and particulate fractions of temporal cortex from bipolar disorder subjects compared with controls. These findings suggest that higher cyclic AMP-dependent protein kinase activity in bipolar disorder brain may be associated with a reduction of regulatory subunits of this enzyme, reflecting a possible adaptive response of this transducing enzyme to increased cyclic AMP signaling in this disorder.     

Cyclic guanosine 3',5'-monophosphate and phosphodiesterase activity in mitogen-stimulated human lymphocytes.

The cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase from human leukemic lymphocytes differes from the normal cell enzyme in having a much higher activity and a loss of inhibition by cyclic guanosine 3′,5′-monophosphate (cyclic GMP). In an effort to determine the mechanism of these alterations, we have studied this enzyme in a model system, lectin-stimulated normal human lymphocytes. Following stimulation of cells with concanavalin A (con A) the enzyme activity gradually becomes altered, until it fully resembles the phosphodiesterase found in leukemic lymphocytes. The changes in the enzyme parallel cell proliferation as measured by increases in thymidine incorporation into DNA. The addition of a guanylate cyclase inhibitor preparation from the bitter melon prevents both the changes in the phosphodiesterase and the thymidine incorporation into DNA. This blockage can be partially reversed by addition of 8-bromo cyclic guanosine 3′,5′-monophosphate (8-bromo cyclic GMP) to the con A-stimulated normal lymphocytes. These results indicate a possible role of cyclic GMP in a growth related alteration of cyclic AMP phosphodiesterase.     

In Vitro Slow Fluctuation in Horseradish Peroxidase Activity

In vitro slow fluctuations in the level of horseradish peroxidase activity were observed in long-range experiments (72–144 h). Besides random fluctuations, regular slow oscillatory patterns with period lengths ranging from 10.0 to 39.0 h were detected by statistical analysis. The possibility that these oscillations in enzyme activity could have reflected changes in the physical environment of the experimental setup has been thoroughly examined and ruled out. Periodic exposition of the enzyme solution, otherwise kept in darkness, to blue light illumination was shown to influence the period of the oscillations. The changes in enzyme activity were correlated with a modification of the Michaelis constant estimated using guaiacol as substrate. This result was confirmed by the action of chemical modifiers of the enzyme, such as ferulic acid and rutin. It is thought that the observed oscillations in horseradish peroxidase activity are due to spontaneous and specific changes in the tridimensional structure of the enzyme in the thermic reservoir.     

Effect of Cyclic AMP-Dependent Protein Phosphorylating Conditions on the pH-Dependent Activity of Tyrosine Hydroxylase from Beef and Rat Striata

Abstract Tyrosine hydroxylase (TH, EC 1.14.16.2) from beef brain striata was purified 23-fold from an extract of an acetone powder. If this enzyme preparation is treated with a cyclic AMP-dependent protein phosphorylation system, there is a change in the pH dependence of the enzyme activity. The pH optimum at saturating tetrahydrobiopterin (BH₄) concentration is shifted from below pH 6 to about pH 6.7. At pH 7, activation is expressed mainly as an increase in V_max, whereas at pH 6, activation is expressed mainly as a decrease in K_m for the pterin cofactor. Further, even with the control enzyme the K_m for pterin cofactor declines precipitously as the pH is increased from 6 toward neutrality. Similar data were obtained with G-25 Sephadex-treated rat striatal TH. Experiments in which rat striatal synaptosomes were used demonstrated that the in situ activation of TH by phosphorylating conditions is expressed primarily as an increase in the maximum rate of dopamine synthesis. These results indicate that changes in TH activity caused by cyclic AMP-dependent protein phosphorylation will depend to a large extent on the pH of the TH environment.     

In Vitro Slow Fluctuation in Horseradish Peroxidase Activity

In vitro slow fluctuations in the level of horseradish peroxidase activity were observed in long-range experiments (72-144 h). Besides random fluctuations, regular slow oscillatory patterns with period lengths ranging from 10.0 to 39.0 h were detected by statistical analysis. The possibility that these oscillations in enzyme activity could have reflected changes in the physical environment of the experimental setup has been thoroughly examined and ruled out. Periodic exposition of the enzyme solution, otherwise kept in darkness, to blue light illumination was shown to influence the period of the oscillations. The changes in enzyme activity were correlated with a modification of the Michaelis constant estimated using guaiacol as substrate. This result was confirmed by the action of chemical modifiers of the enzyme, such as ferulic acid and rutin. It is thought that the observed oscillations in horseradish peroxidase activity are due to spontaneous and specific changes in the tridimensional structure of the enzyme in the thermic reservoir.     

Cyclic Nucleotide Phosphodiesterase Activity in Bovine Brain Coated Vesicles

Cyclic AMP phosphodiesterase activity in bovine brain coated vesicles displayed a Km of approximately 22 microM for cyclic AMP, a Vmax of 3.2 nmol/min/mg protein, and a Hill coefficient of 1.5, suggesting positive cooperativity. The enzyme activity was stimulated by cyclic GMP with maximal indexes of stimulation ranging between 40 and 300%. Both basal and stimulated phosphodiesterase activities were immunotitrated with polyclonal antibodies against clathrin attached to heat-inactivated, formaldehyde-fixed Staphylococcus aureus cells. The main form of phosphodiesterase activity present in the immunoprecipitated brain coated vesicle preparation also is stimulated by cyclic GMP. The allosteric behavior was modulated by cyclic GMP. All of these properties are typical of type II or cyclic GMP-sensitive phosphodiesterases in addition to their calcium and calmodulin independence. Competition experiments with a series of phosphodiesterase inhibitors, papaverine, 1-methyl-3-isobutylxanthine, and theophylline, showed inhibition of cyclic AMP hydrolysis. Trifluoperazine was inactive at the highest concentration used, 100 microM. These compounds also inhibited the cyclic GMP-stimulated cyclic AMP hydrolysis with trifluoperazine practically inactive. At 5 microM cyclic AMP none of the inhibitors was seen to stimulate the cyclic AMP hydrolytic activity. The presence of an enzyme for the breakdown of cyclic nucleotides in brain coated vesicles may suggest a role for these second messengers in the in vivo functions of this organelle.     

Development of Melatonin Synthesis in Chicken Retina: Regulation of Serotonin N-Acetyltransferase Activity by Light, Circadian Oscillators, and Cyclic AMP

In chicken retinas, melatonin levels and the activity of serotonin N-acetyltransferase (NAT), a key regulatory enzyme of melatonin biosynthesis, are expressed as circadian rhythms with peaks of levels and activity occurring at night. In the present study, NAT activity was examined in retinas of embryonic and posthatch chicks to assess the ontogenic development of regulation of the enzyme by light, circadian oscillators, and the second messenger cyclic AMP. During embryonic development, NAT activity was consistently detectable by embryonic day 6 (E6). Significant light-dark differences were first observed on E20, and increased to a maximum amplitude of sixfold by posthatch day 3 (PH3). Circadian rhythmicity of NAT activity appears to develop at or prior to hatching, as evidenced by day-night differences of activity in constant darkness observed in PH1 chicks that had been exposed to a light-dark cycle in ovo only. NAT activity is regulated by a cyclic AMP-dependent mechanism. Activity was significantly increased by incubating retinas with forskolin or dibutyryl cyclic AMP as early as E7, and seven- to ninefold increases were observed following treatment with these agents on E14. Thus, development of the cyclic AMP-dependent mechanism for increasing NAT activity significantly precedes that of rhythmicity, suggesting that the onset of rhythmicity may be related to the onset of photoreception or development of the circadian oscillator in chick retina.     

Biphasic 24-Hour Variations in Cyclic GMP Accumulation in the Rat Pineal Gland Are Due to Corresponding Changes in the Activity of Cytosolic and Particulate Guanylate Cyclase

Various parameters of the rat pineal gland display a 24-h rhythm. However, nothing is known about possible 24-h variations in cyclic GMP (cGMP) metabolism. In the present study, 24-h variations in pineal gland cGMP accumulation were investigated by determining the increase in cGMP level with and without inhibitors of phosphodiesterase at different time points over a light/dark cycle (12/12 h). Furthermore, the activity of guanylate cyclase (GC) was determined under substrate-saturated conditions regarding the cytosolic and particulate forms of the enzyme. It has been found that cGMP accumulation and GC activity display biphasic 24-h variations with two peaks--one approximately 7 h after lights "on" and the other approximately 7 h after lights "off." The activity of cytosolic GC remains unchanged in the presence of the nitric oxide (NO) synthesis inhibitor N-monomethyl-L-arginine, indicating that 24-h variations in the activity do not reflect changes in the synthesis of the GC stimulator NO.     

Phenylalanine-dependent de novo synthesis of phenylalanine ammonia-lyase from Rhodotorula glutinis

A synthetic medium was developed in which the presence of phenylalanine ammonialyase (PAL) in the yeast Rhodotorula glutinis was dependent on the addition of l-phenylalanine. The appearance of PAL activity occurred during mid- to late log phase regardless of the time of l-phenylalanine introduction into the medium. Maximum levels of PAL activity were followed by a rapid decline in both total and specific activity. These changes were accompanied by comparable fluctuations in PAL antigen levels as measured by rocket immunoelectrophoresis. Proteins of yeast grown in the presences of l-phenylalanine were radiolabeled in vivo with l-[³H]leucine. The labeled protein was immunoprecipitated with anti-PAL serum and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A labeled protein comigrated with subunits of authentic PAL. These data support the hypothesis that de novo synthesis of PAL in R. glutinis is l-phenylalanine dependent.     

Gene silencing of cinnamyl alcohol dehydrogenase in Pinus radiata callus cultures

Xylem-derived Pinus radiata cell cultures, which can be induced to differentiate tracheary elements (TEs), were transformed with an RNAi construct designed to silence cinnamyl alcohol dehydrogenase (CAD), an enzyme involved in the biosynthesis of monolignols. Quantitative enzymatic CAD measurements revealed reduced CAD activity levels in most transclones generated. TEs from transclones with approximately 20% residual CAD activity did not release elevated levels of vanillin, which was derived from coniferyl-aldehyde through a mild alkali treatment. However, the activation of the phenylpropanoid pathway in transclones with approximately 20% residual CAD activity through the application of non-physiological concentrations of sucrose and l-phenylalanine produced phenotypic changes. The accumulation of metabolites such as dihydroconiferyl-alcohol (DHCA), which also accumulates in the P. taeda CAD mutant cad-n1, was observed. These results indicate that a substantial reduction in CAD activity is necessary for this enzyme to become a rate-limiting step in lignin biosynthesis in conifers such as P. radiata and confirm that transformable P. radiata callus cultures can be useful to investigate the function of xylogenesis-related genes in conifers.     

Diurnal variation of HMG-CoA reductase activity in rat liver peroxisomes

The specific activity of hepatic microsomal and peroxisomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) was determined at different times during a 24 hour cycle from cholestyramine treated rats. The microsomal HMG-CoA reductase activity displayed a peak at D-6 (6th hour of the dark cycle) as previously reported, whereas, the peroxisomal HMG-CoA reductase activity was the highest at L-2 (2nd hour of the light cycle). Immunoblots of the peroxisomal HMG-CoA reductase suggest that the increase in enzyme activity at L-2 is due to changes in enzyme mass. The different cyclic variations observed in microsomal and peroxisomal HMG-CoA reductase activity may suggest different mechanisms of regulation.     

Effects of inhibition and repression on the utilization of substrates by heterogeneous bacterial communities

This investigation attempts to evaluate to what extent enzyme inhibition and repression by metabolites, indigenous to the cell, are significant phenomena in natural microbial communities. Three case histories of the kinetics of substrate utilization and growth in multisubstrate media by heterogeneous bacterial populations are presented: (i) concurrent substrate utilization and growth on both substrates simultaneously (glucose plus benzoate); (ii) sequential substrate elimination accompanied by diauxic growth as a result of inhibition of enzyme activity (glucose plus galactose); (iii) sequential substrate utilization accompanied by diauxic growth caused by repression of enzyme formation (glucose plus l-phenylalanine, benzoate plus l-phenylalanine). It is shown that enzyme inhibition was observed in two-substrate media as well as in multisubstrate media and was maintained at low substrate concentrations (few milligrams per liter). A special attempt has been made to maintain the diversity of the experimental microbial population during the adaptation and enrichment period. All substrates were determined with sensitive analytical methods specific for the individual substrates. The results obtained confirm that catabolite repression and the resulting sequential substrate utilization are observed in heterogeneous bacterial populations.     

Molecular design of histone deacetylase inhibitors by aromatic ring shifting in chlamydocin framework

Chlamydocin, a cyclic tetrapeptide containing aminoisobutyric acid (Aib), l-phenylalanine (l-Phe), d-proline (d-Pro), and a unique amino acid l-2-amino-8-oxo-9,10-epoxydecanoic acid, inhibits the histone deacetylases (HDACs), a class of enzymes, which play important roles in regulation of gene expression. Sulfur containing amino acids can also inhibit potently, so zinc ligand, such as sulfhydryl group connected with a linker to the so-called capping group, corresponding to cyclic tetrapeptide framework in case of chlamydocin is supposed to interact with the surface of HDAC molecule. Various changes in amino acid residues in chlamydocin may afford specific inhibitors toward HDAC paralogs. To find out specific inhibitors, we focused on benzene ring of l-Phe in chlamydocin framework to shift to various parts of cyclic tetrapeptide. We prepared and introduced several aromatic amino acids into the cyclic tetrapeptides. By evaluating inhibitory activity of these macrocyclic peptides against HDACs, we could find potent inhibitors by shifting the aromatic ring to the Aib site.     

Effect of epinephrine and insulin on adenosine 3'5'-cyclic monophosphate--dependent protein kinase in human skeletal muscle in vivo.

Cyclic AMP dependent protein kinase has beeen identified in human skeletal muscle tissue. In crude muscle extracts the enzyme was 3--5 fold activated by cyclic AMP. The cyclic AMP-dependent activity (corresponding to the inactive holoenzyme) was completely inhibited by the heat stable inhibitor of protein kinase. Reciprocal changes of the cyclic AMP-dependent activity in skeletal muscle were observed after administration of epinephrine and insulin in vivo. Infusion of epinephrine in healthy volunteers increased the level of cyclic AMP and decreased the activity of the cyclic AMP-depenent form (i.e. the inactive form) of protein kinase. These changes were reversible after cessation of epinephrine administration. The results are consistent with an activation of protein kinase in vivo due to an epinephrine mediated increase of the concentration of cyclic AMP. I.v. injection of insulin had the opposite effect on the enzyme in skeletal muscle, leading to increased activity of the cyclic AMP-dependent form of protein kinase. Insulin had no effect on the level of cyclic AMP, but promoted a transient increase of cyclic GMP 1 min. after insulin injection. The effect by insulin on protein kinase cannot be related to the level of cyclic AMP or cyclic GMP.