Aromatic l‐amino acid decarboxylase phosphorylation and activation by PKGIαin vitro

Soluble guanylyl cyclase (sGC) is the major physiological receptor for nitric oxide (NO) throughout the central nervous system. Three different subunits form the α₁/β₁ and α₂/β₁ heterodimeric enzymes that catalyze the reaction of GTP to the second messenger cGMP. Both forms contain a prosthetic heme group which binds NO and mediates activation by NO. A number of studies have shown that NO/cGMP signaling plays a major role in neuronal cell differentiation during development of the central nervous system. In the present work, we studied regulation and expression of sGC in brain of rats during postnatal development using biochemical methods. We consistently observed a surprising decrease in cerebral NO sensitive enzyme activity in adult animals in spite of stable expression of sGC subunits. Total hemoprotein heme content was decreased in cerebrum of adult animals, likely because of an increase in heme oxygenase activity. But the loss of sGC activity was not simply because of heme loss in intact heterodimeric enzymes. This was shown by enzyme activity determinations with cinaciguat which can be used to test heme occupancy in intact heterodimers. A reduction in heterodimerization in cerebrum of adult animals was demonstrated by co‐precipitation analysis of sGC subunits. This explained the observed decrease in NO sensitive guanylyl cyclase activity in cerebrum of adult animals. We conclude that differing efficiencies in heterodimer formation may be an important reason for the lack of correlation between sGC protein expression and sGC activity that has been described previously. We suggest that heterodimerization of sGC is a regulated process that changes during cerebral postnatal development because of still unknown signaling mechanisms.     

Pharmacology of several aromatic amino acid decarboxylase inhibitors]

D,L-beta-(3,4-dihydroxyphenyl)lactic acid (I), D,L-beta-(5-hydroxyindolyl-3)lactic acid (II), and L-alpha-methyl-DOPA (III) inhibited the aromatic amino acid decarboxylase (AAAD) competitively. In difference from the compound III, I and II were not AAAD substrates. Compound II selectively suppressed decarboxylation of L-5-hydroxytryptophane. Compounds I and III potentiated the excitation caused in mice by L-DOPA and failed to influence the excitation due to L-5-hudroxytryptophane (L-5-HTP). Compound II attenuated the excitation caused by L-DOPA and L-5-HTP. Pyridoxine hydrochloride and pyridoxalphosphate attenated the excitation caused by L-DOPA and L-5-HTP. Compounds I and III eliminated this action of vitamins B6.     

Conformations of helical Aib peptides containing a pair of l‐amino acid and d‐amino acid

A pair of l ‐leucine (l ‐Leu) and d ‐leucine (d ‐Leu) was incorporated into α‐aminoisobutyric acid (Aib) peptide segments. The dominant conformations of four hexapeptides, Boc‐l ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1a), Boc‐d ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1b), Boc‐Aib‐Aib‐l ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2a), and Boc‐Aib‐Aib‐d ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2b), were investigated by IR, ¹H NMR, CD spectra, and X‐ray crystallographic analysis. All peptides 1a,b and 2a,b formed 3₁₀‐helical structures in solution. X‐ray crystallographic analysis revealed that right‐handed (P) 3₁₀‐helices were present in 1a and 1b and a mixture of right‐handed (P) and left‐handed (M) 3₁₀‐helices was present in 2b in their crystalline states. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Alpha-synuclein inhibits aromatic amino acid decarboxylase activity in dopaminergic cells

Alpha-synuclein is a presynaptic protein strongly implicated in Parkinson's disease (PD). Because dopamine neurons are invariably compromised during pathogenesis in PD, we have been exploring the functions of alpha-synuclein with particular relevance to dopaminergic neuronal cells. We previously discovered reduced tyrosine hydroxylase (TH) activity and minimal dopamine synthesis in stably-transfected MN9D cells overexpressing either wild-type or A53T mutant (alanine to threonine at amino acid 53) alpha-synuclein. TH, the rate-limiting enzyme in dopamine synthesis, converts tyrosine to l-dihydroxyphenylalanine (L-DOPA), which is then converted to dopamine by the enzyme, aromatic amino acid decarboxylase (AADC). We confirmed an interaction between alpha-synuclein and AADC in striatum. We then sought to determine whether wild-type or A53T mutant alpha-synuclein might have affected AADC activity in dopaminergic cells. Using HPLC with electrochemical detection, we measured dopamine and related catechols after L-DOPA treatments to bypass the TH step. We discovered that while alpha-synuclein did not reduce AADC protein levels, it significantly reduced AADC activity and phosphorylation in our cells. These novel findings further support a role for alpha-synuclein in dopamine homeostasis and may explain, at least in part, the selective vulnerability of dopamine neurons that occurs in PD.     

Redesign of LAOBP to bind novel l‐amino acid ligands

Computational protein design is still a challenge for advancing structure‐function relationships. While recent advances in this field are promising, more information for genuine predictions is needed. Here, we discuss different approaches applied to install novel glutamine (Gln) binding into the Lysine/Arginine/Ornithine binding protein (LAOBP) from Salmonella typhimurium. We studied the ligand binding behavior of two mutants: a binding pocket grafting design based on a structural superposition of LAOBP to the Gln binding protein QBP from Escherichia coli and a design based on statistical coupled positions. The latter showed the ability to bind Gln even though the protein was not very stable. Comparison of both approaches highlighted a nonconservative shared point mutation between LAOBP_graft and LAOBP_sca. This context dependent L117K mutation in LAOBP turned out to be sufficient for introducing Gln binding, as confirmed by different experimental techniques. Moreover, the crystal structure of LAOBP_L117K in complex with its ligand is reported.     

Uncharged tRNA-phosphofructokinase interaction in amino acid deficiency

Summary When the tRNA of mammalian cells is incompletely charged due to amino acid deficiency or by analogs which cannot be activated, many metabolic events become limited. This rapid demise of cell function appears to be due to the inhibition of phosphofructokinase (PFK) by uncharged tRNA (FEBS Lett 302: 113 (1992)). Charged tRNA has been shown to be sequestered within the protein synthetic machinery, (Negrutskii, B. S. and Deutscher, M. P. (1992) Proc Natl Acad Sci USA 89: 3601) and would therefore be removed from an inhibitory role. Besides the direct demonstration that tRNA inhibits PFK in an assay regarded as indicative of its control mechanism, several reports in the literature support this model. These include 1) The rapid onset of inhibition of glycolysis and glucose uptake by intact cells upon amino acid deficiency and the similar lesion at the 43S ribosomal subunit on glucose or amino acid deprivation. 2) The recognition that unusually high concentrations of cAMP required to stimulate protein synthesis in energy depleted or gel filtered lysates correlates with its action on PFK as an analog of the positive effector, adenosine-5-monophosphate. 3) The often repeated observation that the product of PFK activity, fructose-1,6-diphosphate, is a stimulant of protein synthesis (see Jackson, R. J., et al. (1983) Eur J Biochem 131: 289). This diphosphate has been shown to be the proximate effector binding to eIF-2B, the guanine nucleotide exchange factor (Singh, L. P. Arror, A. R. and Wahba, A. J. (1994), FASEB J. 8: 279) which by releasing GDP bound to the inactive GDP: eIF-2 complex, permits the factor to initiate a new peptide chain. The above information supports the view that the block at the G1 restriction point in the cell cycle of normal cells brought about by amino acid deprivation is a result of inhibition of protein synthesis through the phosphofructokinase-uncharged tRNA mechanism. This is consistent with observations in the literature that tumor and transformed cells, which are more resistant to this block (Pardee, A. B., Proc Natl Acad Sci USA 71: 1286–1291 (1974)) have a higher phosphofructokinase activity or higher levels of fructose-1,6-diphosphate.Presented at the 4th International Congress on Amino Acids, Vienna, Austria, August 7–11, 1995. (Amino Acids (1995) 9: 23, Abstract).     

Enhanced dissolution of the substrate d,l‐2‐amino‐Δ2‐thiazoline‐4‐carboxylic acid and enzymatic production of l‐cysteine at high concentrations

l ‐Cysteine is widely used as a precursor in the pharmaceutical, cosmetic, food, and feed additive industries. It has been industrially produced from hydrolysis of human and animal hairs, which is limited for industrial production. At the same time, chemical hydrolysis causes the formation of intractable waste material. Thus, environmentally friendly methods have been developed. A big obstacle of currently available methods is the low substrate solubility leading to poor l ‐cysteine yield. Here, a method for improving the low solubility of the substrate d ,l ‐2‐amino‐Δ²‐thiazoline‐4‐carboxylic acid (d ,l ‐ATC) is presented and the enzymatic reaction at high concentration levels was optimized. The substrate was dissolved in large amounts in aqueous solutions by pH control using salts. d ,l ‐ATC solubility increased with an increasing solution pH due to its enhanced hydrophilicity, which can be achieved by a shift to dissociated carboxylic group (–COO^?). The highest d ,l ‐ATC solubility of 610 mM was obtained at pH 10.5. The maximum l ‐cysteine yield of 250 mM was attained at pH 9.1, which lies between the optimum values for high substrate solubility and reaction rate. The product yield could be increased by more than 10 times compared to those in previous reports, which is industrially meaningful.     

A method for the direct detection of aromatic amino acid decarboxylase in electrophoretic media

Aromatic amino acid decarboxylase is an enzyme of broad specificity which decarboxylates a range of aromatic amino acids including 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, which yield dopamine and serotonin, respectively. The name aromatic amino acid decarboxylase was applied to the enzyme by workers (1–3) who found that, on purification, a single enzyme appeared to be responsible for activities previously ascribed to two separate enzymes. The separate enzymes had been named 5-hydroxy-l-tryptophan carboxylyase (EC 4.1.1.28) and 3,4-dihydroxy-l-phenylalanine carboxylyase (EC 4.1.1.26). More recent papers (4–7) still leave unresolved the question of whether one enzyme or two is responsible for the two activities.     

Identification and characterization of an aromatic amino acid decarboxylase from the filarial nematode, Dirofilaria immitis

A novel secreted aromatic amino acid decarboxylase-like molecule was identified in the excretory/secretory products of L3/L4 larvae as well as in an extract of adult Dirofilaria immitis. The secretion of the enzyme was developmentally regulated. Peak enzyme activities were detected in the culture medium before and after the molting of L3 larvae in vitro. The enzyme was purified from D. immitis adult extracts and the excretory/secretory products of L3/L4 larvae using different chromatographic methods followed by isoelectric focusing and SDS-PAGE. The enzyme has a molecular mass of 48 kDa and a pI of 5.6, and shows a specific enzymatic activity towards the aromatic amino acid substrates phenylalanine, tyrosine and tryptophan. The enzyme's activity did not show an absolute requirement for exogenous pyridoxal-5-phosphate. However, addition of pyridoxal-5-phosphate at 5 microM in the reaction increased the enzyme activity greatly. The enzyme had the ability to catalyze the formation of dopamine from L-dopa. Studies on the effects of inhibitors on the enzyme activity showed that the enzyme was sensitive to Pefabloc and p-chloromercuribenzoic acid, but not to diisopropyl flurophosphate. The Km values of the enzyme for H-Phe-AMC, H-Tyr-AMC and H-Trp-AMC were calculated to be 32.1 microM, 35.1 microM and 29.1 microM, respectively.     

Glutathione plays an essential role in nitric oxide‐mediated iron‐deficiency signaling and iron‐deficiency tolerance in Arabidopsis

Iron (Fe) deficiency is a common agricultural problem that affects both the productivity and nutritional quality of plants. Thus, identifying the key factors involved in the tolerance of Fe deficiency is important. In the present study, the zir1 mutant, which is glutathione deficient, was found to be more sensitive to Fe deficiency than the wild type, and grew poorly in alkaline soil. Other glutathione‐deficient mutants also showed various degrees of sensitivity to Fe‐limited conditions. Interestingly, we found that the glutathione level was increased under Fe deficiency in the wild type. By contrast, blocking glutathione biosynthesis led to increased physiological sensitivity to Fe deficiency. On the other hand, overexpressing glutathione enhanced the tolerance to Fe deficiency. Under Fe‐limited conditions, glutathione‐deficient mutants, zir1, pad2 and cad2 accumulated lower levels of Fe than the wild type. The key genes involved in Fe uptake, including IRT1, FRO2 and FIT, are expressed at low levels in zir1; however, a split‐root experiment suggested that the systemic signals that govern the expression of Fe uptake‐related genes are still active in zir1. Furthermore, we found that zir1 had a lower accumulation of nitric oxide (NO) and NO reservoir S‐nitrosoglutathione (GSNO). Although NO is a signaling molecule involved in the induction of Fe uptake‐related genes during Fe deficiency, the NO‐mediated induction of Fe‐uptake genes is dependent on glutathione supply in the zir1 mutant. These results provide direct evidence that glutathione plays an essential role in Fe‐deficiency tolerance and NO‐mediated Fe‐deficiency signaling in Arabidopsis.     

Study of the conformational profile of selected unnatural amino acid residues derived from l‐phenylalanine

The present work reports the results of a conformational study performed on seven unnatural amino acid residues and on its natural precursor, investigated by means of computational methods at the molecular mechanics level. Amino acid residues selected for the present study are derivatives of l ‐phenylalanine substituted at the α and/or β carbons. This series is composed of different linear analogs, including α‐methyl, β‐methyl and β‐phenyl substituted with different stereochemistry. Analysis of the Ramachandran maps of the corresponding dipeptides in vacuo reveals their conformational preferences, to be used as guidance for the synthesis of constrained peptide analogs with desired conformational propensities. The available conformational space for every dipeptide is also analysed. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Enantioselective resolution of 4‐chloromandelic acid by liquid–liquid extraction using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid

A liquid–liquid extraction resolution of 4‐chloro‐mandelic acid (4‐ClMA) was studied by using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid (2‐Cl‐Z‐AA) as a chiral extractant. Important factors affecting the extraction efficiency were investigated, including the type of chiral extractant, pH value of aqueous phase, initial concentration of chiral extractant in organic phase, initial concentration of 4‐ClMA in aqueous phase, and resolution temperature. It was observed that the concentration of (R)‐4‐ClMA was much higher than that of (S)‐4‐ClMA in organic phase due to a higher stability of the complex formed between (R)‐4‐ClMA and 2‐Cl‐Z‐AA. A separation factor (α) of 3.05 was obtained at 0.02 mol/L 2‐Cl‐Z‐Valine dissolved in dichloromethane, pH of 2.0, concentration of 4‐ClMA of 0.11 mmol/Land T of 296.7K.     

An amino acid code for β‐sheet packing structure

To understand the relationship between protein sequence and structure, this work extends the knob‐socket model in an investigation of β‐sheet packing. Over a comprehensive set of β‐sheet folds, the contacts between residues were used to identify packing cliques: sets of residues that all contact each other. These packing cliques were then classified based on size and contact order. From this analysis, the two types of four‐residue packing cliques necessary to describe β‐sheet packing were characterized. Both occur between two adjacent hydrogen bonded β‐strands. First, defining the secondary structure packing within β‐sheets, the combined socket or XY:HG pocket consists of four residues i, i+2 on one strand and j, j+2 on the other. Second, characterizing the tertiary packing between β‐sheets, the knob‐socket XY:H+B consists of a three‐residue XY:H socket (i, i+2 on one strand and j on the other) packed against a knob B residue (residue k distant in sequence). Depending on the packing depth of the knob B residue, two types of knob‐sockets are found: side‐chain and main‐chain sockets. The amino acid composition of the pockets and knob‐sockets reveal the sequence specificity of β‐sheet packing. For β‐sheet formation, the XY:HG pocket clearly shows sequence specificity of amino acids. For tertiary packing, the XY:H+B side‐chain and main‐chain sockets exhibit distinct amino acid preferences at each position. These relationships define an amino acid code for β‐sheet structure and provide an intuitive topological mapping of β‐sheet packing. Proteins 2014; 82:2128–2140. © 2014 Wiley Periodicals, Inc.     

Inhibitors binding to L-aromatic amino acid decarboxylase

The effect of a number of inhibitors of L-aromatic amino acid decarboxylase activity on the absorption spectrum of the enzyme-bound coenzyme has been studied. It has been observed that the compounds tested, even if devoid of the amino function and therefore unable to form the Schiff base with the coenzyme, modify significantly the enzyme spectrum, indicating their binding to the coenzyme active site. Spectral modifications suggest that at least two kinds of binding of inhibitors to L-aromatic amino acid decarboxylase may occur, depending on their structural features. Moreover, from the spectra obtained at different concentrations of the inhibitors their affinity constants have been determined: data indicate that the cathecol ring gives the largest contribution to the binding, while the presence of the carboxyl group, the aminic group and the aliphatic chain are responsible for a decrease in the binding, which could be relevant for the efficiency of the catalysis.     

Manganese deficiency leads to elevated amino acid pools in citric acid accumulating Aspergillus niger

Free amino acid pools have been investigated in a citric acid accumulating strain of Aspergillus niger during batch growth under manganese sufficient and deficient conditions by means of an improved chromatographic method. Studies on the mycelial content of several nitrogenous compounds under manganese sufficient and deficient conditions showed that manganese deficiency resulted in lower amino acid pool sizes during trophophase and considerable accumulation during idiophase, and in a reduction of the protein and nucleic acid contents. Addition of cycloheximide to mycelia grown with sufficient manganese also caused an elevation of free amino acid pool sizes, thus indicating that impairment of protein synthesis by manganese deficiency is responsible for the observed rise in amino acid concentration. Furthermore it was observed that the manganese deficient mycelia excreted high amounts of all amino acids suggesting that manganese deficiency may also affect membrane permeability.