The dark respiration of Anacystis nidulans: Production of HCN from histidine and oxidation of basic amino acids

The basic amino acids, L-arginine, L-lysine, L-ornithine, and to a lesser extent L-histidine, strongly stimulate the O₂ uptake of cell suspensions of the blue-green alga or cyanobacterium Anacystis nidulans. In the case of L-histidine, the extra O₂ consumption is associated with the formation in vivo of small amounts of HCN, particularly in an atmosphere of O₂. The enzyme responsible for both the stimulated O₂ uptake with the basic amino acids and the formation of HCN from histidine has been isolated and identified as an L-amino acid oxidase specific for the basic amino acids. The purification (15 000-fold) of this enzyme is described. The isolated enzyme is inhibited by o-phenanthroline, which has a similar inhibitory effect on the O₂ uptake of cell suspensions with (and without) added amino acids.The basic amino acid oxidase, which is not inhibited by HCN, can be regarded as an ‘alternate’ oxidase in A. nidulans. An oxidase sensitive to HCN is apparently also operative. At high concentrations of lysine or arginine added HCN can almost double the initial rate of O₂ consumption of cell suspensions. This can be attributed to the inhibition of catalase by HCN. At low concentrations of the amino acids, and with more prolonged incubation time, HCN becomes inhibitory. One interpretation could be that the HCN-sensitive terminal oxidase is also involved in the extra O₂ uptake elicited by the basic amino acids, but other interpretations are possible. The extra O₂ uptake elicited by histidine is almost completely inhibited by HCN, which is consistent with the finding that histidine is a relatively poor substrate for the basic amino acid oxidase.     

A D-amino acid oxidase from Chlorella vulgaris.

A procedure has been developed for the partial purification from Chlorella vulgaris of an enzyme which catalyzes the formation of HCN from D-histidine when supplemented with peroxidase of a metal with redox properties. Some properties of the enzyme are described. Evidence is presented that the catalytic activity for HCN formation is associated with a capacity for catalyzing the oxidation of a wide variety of D-amino acids. With D-leucine, the best substrate for O2 consumption, 1 mol of ammonia is formed for half a mol of O2 consumed in the presence of catalase. An inactive apoenzyme can be obtained by acid ammonium sulfate precipitation, and reactivated by added FAD. On the basis of these criteria, the Chlorella enzyme can be classified as a D-amino acid oxidase (EC 1.4.3.3). Kidney D-amino acid oxidase and snake venom L-amino acid oxidase, which likewise form HCN from histidine on supplementation with peroxidase, have been compared with the Chlorella D-amino acid oxidase. The capacity of these enzymes for causing HCN formation from histidine is about proportional to their ability to catalyze the oxidation of histidine.     

Cyanide formation from histidine in Chlorella. A general reaction of aromatic amino acids catalyzed by amino acid oxidase systems.

The formation of HCN from D-histidine in Chlorella vulgaris extracts is shown to be due to the combined action of a soluble protein and a particulate component. Either horse-radish peroxidase (EC 1.11.1.7) or a metal ion with redox properties can be substituted for the particulate component. Ions of manganese and vanadium are especially effective, as are o-phenanthroline complexes of iron. Cobalt ions are less active. The D-amino acid oxidase (EC 1.4.3.3) from kidney and the L-amino acid oxidase (EC 1.4.3.2) from snake venom likewise cause HCN production from histidine when supplemented with the particulate preparation from Chlorella or with peroxidase or with a redox metal ion. The stereospecificity of the amino acid oxidase determines which of the two stereoisomers of histidine is active as an HCN precursor. Though histidine is the best substrate for HCN production, other naturally occurring aromatic amino acids (viz. tyrosine, phenylalanine and tryptophan) can also serve as HCN precursors with these enzyme systems. The relative effectiveness of each substrate varies with the amino acid oxidase enzyme and with the supplement. With respect to this latter property, the particulate preparation from Chlorella behaves more like a metal ion than like peroxidase.     

Studies on the production and assessment of experimental histidinemia in the rat.

Intraperitoneal administration to rats of D- or DL-alpha-hydrazinoimidazolylpropionic acid was found to produce a substantial inactivation of hepatic histidine ammonia-lyase (EC 4.3.1.3) in vivo. Proportional to this loss in enzyme activity was an impairment of the ability of treated rats to oxidize L-[ring-2-14C] histidine to 14CO2. Rats in which hepatic histidine ammonia-lyase activity was either depressed by DL-hydrazinoimidazolylproprionic acid injection or elevated by feeding a high protein diet displayed proportionately altered rates of 3H2O release into plasma water following L-[3-3H] histidine administration. Plasma L-histidine clearance following loading with this amino acid was similarly affected by these treatments. Administration of DL-alphal-hydrazinoimisazolylproprionic acid to rats was also found to inactivate non-specifically pyridoxal 5-phosphate enzymes in vivo; pyridoxine injection was found to reverse the DL-alpha-hydrazinoimidazolylproprionic acid-induced inactivation of hepatic aspartate aminotransferase (EC 2.6.1.1) in vivo, but not that of hepatic histidine ammonia-lyase. These findings demonstrate that histidine ammonia-lyase is the rate-limiting factor in L-histidine degradation in the rat. The potential usefulness of DL-hydrazinoimidazolylproprionic acid in the production of an animal model for histidinemia (hereditary histidine ammonia-lyase deficiency) is discussed.     

L-histidine utilization in Aspergillus nidulans.

Histidase activity rather than uptake of L-histidine is the limiting factor for the utilization of histidine as the sole nitrogen source for Aspergillus nidulans. Histidine cannot act as the sole carbon source, and evidence is presented indicating that this is attributable to an inability to convert histidine to L-glutamate in vivo. It has been shown that this fungus lacks an active urocanase enzyme and that histidine is quantitatively converted to urocanate, which accumulates in the extracellular medium. The use of histidine as a nitrogen source is regulated by nitrogen metabolite repression control of histidase synthesis. In addition, evidence for a requirement for a carbon source for histidase synthesis and for a minor form of control by nitrate is presented. The activity of the histidase enzyme is inhibited by micromolar concentrations of the product urocanate and by physiological levels of L-glutamate and L-glutamine.     

Mechanism of L-serine oxidation in Entamoeba histolytica.

1. The enzymatic mechanism of oxygen uptake elicited by L-serine in axenically cultivated trophozoites of Entamoeba histolytica was investigated. 2. Of 22 amino acids examined, only L-serine stimulated oxygen consumption by intact and disrupted amoebae. 3. Pyruvate, a product of serine metabolism, also stimulated oxygen consumption in the amoebae. 4. Characterization of the oxygen uptake elicited by both L-serine and pyruvate, and analysis of the products of L-serine metabolism indicate that the amino acid is first converted to pyruvate. 5. L-Serine dehydratase, which catalyzes the deamination of serine to pyruvate, was detected primarily in the soluble fraction of the amoebae. D-Serine potently inhibited the enzyme, as well as oxygen uptake in the presence of L-serine but not in the presence of pyruvate. 6. The pyruvate formed is oxidized, at least in part, by a novel pyruvate oxidase involving the uptake of molecular oxygen.     

Characterization of a phagocyte cytochrome b558 91-kilodalton subunit functional domain: identification of peptide sequence and amino acids essential for activity.

The phagocyte NADPH oxidase is a multicomponent membrane-bound electron transport chain that catalyzes the reduction of O2 to superoxide. Cytochrome b558, the terminal electron donor to O2, is an integral membrane heterodimer containing 91- and 22-kDa subunits (gp91-phox and p22-phox, respectively). Synthetic peptides, whose amino acid sequences correspond to a gp91-phox carboxyl-terminal domain, inhibit superoxide production by blocking assembly of the oxidase from membrane and cytosol components. In this study, we examined the amino acid sequence requirements of a series of synthetic truncated gp91-phox peptides for inhibition of human neutrophil NADPH oxidase activation. RGVHFIF, corresponding to gp91-phox residues 559-565, was the minimum sequence capable of inhibiting superoxide generation. Contributions of individual amino acids to overall RGVHFIF inhibitory activity were determined by comparing the abilities of alanine-substituted RGVHFIF peptides to inhibit superoxide production. Substitution of alanine for arginine, valine, isoleucine, or either of the phenylalanines (but not glycine or histidine) within RGVHFIF resulted in loss of inhibitory activity. Synthetic gp91-phox carboxyl-terminal peptides are likely to be competitive inhibitors of the corresponding carboxyl-terminal domain of native gp91-phox by virtue of amino acid identity. We conclude that properties of arginine valine, isoleucine, and phenylalanine side chains within an RGVHFIF-containing domain of gp91-phox contribute significantly to cytochrome b558-mediated activation of the oxidase.     

pH Dependence of Histidine Affinity for Blood-Brain Barrier Carrier Transport Systems for Neutral and Cationic Amino Acids

The effects of pH (3.5-7.5) on the brain uptake of histidine by the blood-brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single-pass carotid injection technique. Cationic amino acid ( [14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ( [14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.     

Methionine transport in S37 cells. Substrate-dependent function of amino acid transport system A in exchange processes.

Methionine had been observed to interact with two principal transport systems for amino acids in mammalian cells, the A and L systems. The present study of methionine transport and of exchange processes through system A arose in the course of a study to define the specificity of a transinhibition effect caused by cysteine. Methionine uptake through two transport systems in the S37 cell was confirmed by the occurrence of a biphasic double-reciprocal plot for labeled methionine uptake. Preloading cells with methionine stimulated labeled histidine uptake through systems A and L. Efflux of labeled methionine from cells was stimulated by histidine in a biphasic manner, so that bothe systems A and L can be used for exchange when methionine is the intracellular amino acid. Aminocycloheptanecarboxylic acid elicited exchange efflux of labeled methionine only through system L. ALPHA-Aminoisobutyric acid and N-methyl-alpha-aminoisobutyric acid both stimulated efflux of labeled N-methyl-alpha-aminoisobutyric acid from S37 cells. These findings are interpreted a showing that transport system A is capable of functioning as an exchange system depending upon the identity of intracellular and extracellular substrates available.     

Polymerase-catalyzed synthesis of DNA from phosphoramidate conjugates of deoxynucleotides and amino acids

Some selected amino acids, in particular L-aspartic acid (L-Asp) and L-histidine (L-His), can function as leaving group during polymerase-catalyzed incorporation of deoxyadenosine monophosphate (dAMP) in DNA. Although L-Asp-dAMP and L-His-dAMP bind, most probably, in a different way in the active site of the enzyme, aspartic acid and histidine can be considered as mimics of the pyrophosphate moiety of deoxyadenosine triphosphate. L-Aspartic acid is more efficient than D-aspartic acid as leaving group. Such P-N conjugates of amino acids and deoxynucleotides provide a novel experimental ground for diversifying nucleic acid metabolism in the field of synthetic biology.     

l-amino acid oxidases of Proteus rettgeri.

Proteus rettgeri has been found to contain two separable 1-amino acid oxidases. Both enzymes are particulate in nature, neither being ribosomal bound. One of these enzymes appears to have broad specificity, being active toward monoaminomonocarboxylic, imino, aromatic, sulfur-containing, and beta-hydroxyamino acids. The other enzyme has more limited specificity, catalyzing the oxidative deamination of the basic amino acids and citrulline. The affinity of this oxidase for the various substrates at pH 7.6 in decreasing order is arginine, histidine, ornithine, citrulline, and lysine. This enzyme has a particularly high affinity for arginine (Km equal to 0.27 mM), and anomalous kinetics are observed with increasing substrate concentrations. When concentrations of arginine greater than 1.0mM were added to the reaction containing histidine, imidazole pyruvate formation was completely inhibited.     

Effects of Mn2+, Ca2+ and chlorpromazine on photosystem II of Anacystis nidulans. An attempt to establish a functional relationship of amino acid oxidase to photosystem II

Washing with EDTA changes the specificity of Anacystis nidulans particles having photosystem II activities for activation by cations. A specific requirement for Mn2+ and a somewhat lower specificity for Ca2+ can be demonstrated in the EDTA-washed particles. Both ions must be added to reconstitute the system evolving O2 in the light. EDTA-washed particles retain the L-amino acid oxidase with high specificity for the basic L-amino acids [Pistorius, E. K. and Voss, H. (1980) Biochem. Biophys. Acta, 611, 227-240] as well as the ability to reduce 2,6-dichloroindophenol with diphenylcarbazide as a donor in the light. The latter reaction which does not require added cations, can be inhibited by chlorpromazine, and this inhibition can be partially relieved by Ca2+ ions. Evidence is also presented that the L-amino-acid oxidase is inhibited by chlorpromazine, and this inhibition can be relieved by L-arginine in much the same way as the inhibition of the enzyme by Ca2+ ions can be relieved by L-arginine. The data are compatible with, but do not prove, an involvement of the L-amino-acid oxidase in the redox reactions of photosystem II of A. nidulans.     

Purification and characterization of rat liver mitochondrial phenylalanine pyruvate aminotransferase.

Phenylalanine pyruvate aminotransferase in rat liver was found in both the mitochondrial and supernatant fractions. Phenylalanine pyruvate aminotransferase was purified from rat liver mitochondria. The purified enzyme was specific for pyruvate, exhibiting no activity with 2-oxoglutarate as aminoacceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order of activity: L-phenylalanine > L-tyrosine > L-histidine > 3,4-dihydroxy-DL-phenylalanine. Very little activity was observed with L-tryptophan and 5-hydroxy-L-tryptophan. The apparent Km values for L-phenylalanine and L-histidine were 2.6 mM and 2.7 mM, respectively. The Km values for pyruvate were 5.0 mM and 1.5 mM with phenylalanine and histidine as amino donors, respectively. The pH optimum was near 9.0. Sucrose density gradient centrifugation gave a molecular weight of approximately 68,000. On the basis of subcellular distributions, substrate specificities, substrate inhibition, pH optima, polyacrylamide gel electrophoresis and some other properties, it was suggested that mitochondrial phenylalanine pyruvate aminotransferase was identical with mitochondrial histidine pyruvate aminotransferase.     

Transport of basic amino acids in Candida albicans

In Candida albicans, ATCC 46977, transport of basic amino acids is mediated by two systems (S1 and S2). Kinetic data and competitive inhibition studies of the different systems showed that transport of L-lysine, L-arginine and L-histidine have distinct specificities. System S1 of L-lysine and L-arginine was highly specific for the respective single basic amino acid. However, S2 of L-lysine and S1 of L-histidine were shown to be specific systems for most of basic amino acids. S2 of L-arginine was different from S2 of L-lysine and S1 of L-histidine. The effect of a thiol reagent, N-ethylmalemide, revealed that S2 of L-lysine and S1 of L-histidine were sensitive to this reagent, while all other systems were insensitive. The transport activity of different systems of L-lysine, L-arginine and L-histidine was followed during the growth of C. albicans. It was observed that different basic amino-acid systems have maximum activity during different stages of C. albicans growth.     

Dietary supplements of mixtures of indispensable amino acids lacking threonine, phenylalanine or histidine increase the activity of hepatic threonine dehydrogenase, phenylalanine hydroxylase or histidase, respectively, and prevent growth depressions in chicks caused by dietary excesses of threonine, phenylalanine, or histidine*

Experiments were carried out to determine whether the addition of a mixture of indispensable amino acids (IAA) lacking in threonine, phenylalanine or histidine, respectively, to a nutritionally complete diet would increase the hepatic activities of the rate-limiting enzymes for catabolism of threonine, phenylalanine or histidine and prevent the adverse effects of the amino acid on growth when the dietary level of the amino acid is excessive. Week old Leghorn chicks were fed semi-purified diets containing 19% crude protein to which were added no IAA supplement or 10% crude protein from an IAA mix and 5 graded levels of either L-threonine, L-phenylalanine or L-histidine in a 2 x 5 factorial arrangement of treatments. Each amino acid was investigated in a separate experiment involving four replicate pens (seven chicks each) per diet. Weight gains and feed consumptions were determined on the fourteenth day of each experiment. The groups receiving no excess, and 1.0% or 2.0% excesses of amino acids were sampled on the fifteenth day for enzyme activities and plasma amino acid concentrations. Weight gain and/or feed consumption were lower, and plasma concentrations of threonine, phenylalanine and histidine were higher, in chicks receiving 1.5 to 2.0% dietary additions of threonine, phenylalanine, and histidine, respectively, than in chicks that did not receive these amino acids. Chicks that received the amino acids in diets that also contained the IAA supplement had better growth and feed consumption, lower plasma concentrations of threonine, phenylalanine or histidine, higher plasma concentrations of other indispensable amino acids, and higher activities of threonine dehydrogenase, phenylalanine hydroxylase, and histidase than chicks receiving excess amino acids in the absence of IAA supplements. We conclude that the dietary level of protein, not the dietary level of individual amino acids, is the primary determinant of the activity of amino acid degrading enzymes in liver. The increased activity of these enzymes may be the mechanism by which dietary protein alleviates the adverse effects of excessive levels of individual amino acids.     

L-Histidine production by histidase-less regulatory mutants of Serratia marcescens constructed by transduction.

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.     

L-Histidine production by histidase-less regulatory mutants of Serratia marcescens constructed by transduction.

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.     

Mechanism of amino Acid uptake by sugarcane suspension cells

The amino acid carriers in sugarcane suspension cells were characterized for amino acid specificity and the stoichiometry of proton and potassium flux during amino acid transport.

Amino acid transport by sugarcane cells is dependent upon three distinct transport systems. One system is specific for neutral amino acids and transports all neutral amino acids including glutamine, asparagine, and histidine. The uptake of neutral amino acids is coupled to the uptake of one proton per amino acid; one potassium ion leaves the cells for charge compensation. Histidine is only taken up in the neutral form so that deprotonation of the charged imidazole nitrogen has to occur prior to uptake. The basic amino acids are transported by another system as uniport with charge-compensating efflux of protons and potassium. The acidic amino acids are transported by a third system. Acidic amino acids bind to the transport site only if the distal carboxyl group is in the dissociated form (i.e. if the acidic amino acid is anionic). Two protons are withdrawn from the medium and one potassium leaves the cell for charge compensation during the uptake of acid amino acids. Common to all three uptake systems is a monovalent positively charged amino acidproton carrier complex at the transport site.

     

A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.     

Stimulation of amino acid accumulation in neuroblastoma and astrocytoma cells byl-histidine

Uptake of amino acids by cultured neuroblastoma and astrocytoma cells was studied in the presence and absence ofl-histidine. Intracellularly accumulated histidine was assumed to induce accumulation of radioactively labeled amino acids from medium by means of exchange transport. Neuroblastoma cells accumulated more histidine than astrocytoma cells and were more sensitive to the enhancement of the uptake of other large neutral amino acids by histidine. Histidine also increased glutamic acid uptake in astrocytoma cells, but reduced it in neuroblastoma cells. The greatest differences between the cell lines in amino acid uptake without histidine were found with acidic amino acids (astrocytoma cells accumulated them more than neuroblastoma cells) and with taurine (the reverse was found). The uptake and exchange mechanisms for some neutral and acidic amino acids may thus be dissimilar in the plasma membranes of cultured cells of neuronal and glial origin.