Synthesis of sugar-amino acid-nucleosides as potential glycosyltransferase inhibitors

Sugar-amino acid-nucleosides (SAAN) were synthesized to mimic glycosyl nucleotide donors based on the hypothesis that a basic amino acid may interact with carboxylate groups of the enzyme in a manner similar to the diphosphate metal ion complex. C-Glycoside analogues of the d-galactopyranose or l-arabinofuranose ring systems, and four amino acids (lysine, glutamine, tryptophan, and histidine), were chosen for this study. The targets were synthesized and tested against GlfT2, a galactofuranosyltransferase essential for cell wall galactan biosynthesis in Mycobacterium tuberculosis. The inhibition assay showed that analogues containing histidine and tryptophan are moderate inhibitors of GlfT2.     

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and γ-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra × maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and γ-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.     

Utilization and requirements of amino acids by a cell line derived from the butterfly Papilio xuthus

The media, in which a butterfly cell line (Px 58), derived from pharate adult ovaries of Papilio xuthus cultured for 8 days, were analysed to examine the changes in free amino acids in the medium during cultivation. Beta-alanine, arginine, glycine, histidine, lysine, phenylalanine, proline, serine, and tryptophan did not change markedly. Asparagine, aspartic acid, cystine, glutamine, isoleucine, leucine, methionine, threonine, tyrosine, and valine decreased to some extent with culturing. Alpha-alanine increased markedly, and glutamic acid did so to a lesser extent. Requirements of amino acids by the cell line were examined by deleting amino acids one at a time. Deletion of alpha-alanine, beta-alanine, asparagine, glutamic acid, glycine, and phenylalanine did not cause deterioration of the cell. These amino acids were thought to be non-essential or required only a little. Deletion of other amino acids impaired the cell growth severely. These amino acids would appear to be essential for growth of the Px 58 cell line.     

Regulation of exocellular protease in Neurospora crassa: induction and repression under conditions of nitrogen starvation.

Exponential-phase cells of Neurospora crassa require the continued presence of a protein inducer and nitrogen starvation to induce exocellular protease under conditions where protein is the sole nitrogen source. The nature of the protein inducer appears relatively unimportant, since both soluble proteins (e.g., myoglobin) and insoluble proteins (e.g., corn zein) will effect induction. Nonstarved cells of N. crassa appear to have small nitrogen pools, since nitrogen starvation of exponential cells prior to transfer into a medium where protein is the sole nitrogen source effects starvation-time-dependent decreases in protease biosynthesis. Ammonium ion represses protease synthesis, with apparent specificity at low concentrations. The amino acids arginine, tryptophan, and threonine effect repression of protease biosynthesis under conditions of nitrogen starvation. Under conditions of sulfur starvation, the amino acids cysteine, methionine, and cystine repress protease biosynthesis. In carbon-starved cells, all of the above amino acids, plus histidine, isoleucine, leucine, lysine, phenylalanine, and valine, effect repression. Examination of amino acid pools formed when cells are grown on protein as the sole nitrogen source demonstrated that the amino acids which repress protease biosynthesis under conditions where protein is the sole carbon source accumulate in significant amounts during the course of protease induction, with kinetics consonant with the induction process.     

Cross-Pathway Regulation: Histidine-Mediated Control of Histidine, Tryptophan, and Arginine Biosynthetic Enzymes in Neurospora crassa

In Neurospora crassa, histidine starvation of histidine mutants resulted in derepression of histidine, tryptophan, and arginine biosynthetic enzymes. The same tripartite derepression occurred in wild-type strain 74A when it was grown in medium supplemented with 3-amino-1,2,4-triazole, an inhibitor of histidine biosynthesis. Histidine-mediated derepression of tryptophan and arginine biosynthetic enzymes was not due to a lowered intracellular concentration of tryptophan or arginine, respectively. A discussion of possible mechanisms and of similar studies in prokaryotic and eukaryotic organisms is presented.     

The Bacillus subtilis Chemoreceptor McpC Senses Multiple Ligands Using Two Discrete Mechanisms

Bacillus subtilis can perform chemotaxis toward all 20 l-amino acids normally found in proteins. Loss of a single chemoreceptor, McpC, was previously found to reduce chemotaxis to 19 of these amino acids. In this study, we investigated the amino acid-sensing mechanism of McpC. We show that McpC alone can support chemotaxis to 17 of these amino acids to varying degrees. Eleven amino acids were found to directly bind the amino-terminal sensing domain of McpC in vitro. Sequence analysis indicates that the McpC sensing domain exhibits a dual Per-Arnt-Sim (PAS) domain structure. Using this structure as a guide, we were able to isolate mutants that suggest that four amino acids (arginine, glutamine, lysine, and methionine) are sensed by an indirect mechanism. We identified four candidate binding lipoproteins associated with amino acid transporters that may function in indirect sensing: ArtP, GlnH, MetQ, and YckB. ArtP was found to bind arginine and lysine; GlnH, glutamine; MetQ, methionine; and YckB, tryptophan. In addition, we found that ArtP, MetQ, and YckB bind the sensing domain of McpC, suggesting that the three participate in the indirect sensing of arginine, lysine, methionine, and possibly tryptophan as well. Taken together, these results further our understanding of amino acid chemotaxis in B. subtilis and gain insight into how a single chemoreceptor is able to sense many amino acids.     

Identification of amino acid residues essential to the activity of lyase CpcT1 from Nostoc sp. PCC7120

The phycocyanin lyase CpcT1 (encoded by gene all5339) and lyase CpcS1 (encoded by gene alr0617) are capable of catalyzing the phycocyanobilin (PCB) covalently bound to the different sites of phycocyanin's and phycoerythrocyanin's β subunits, respectively. Lyase CpcS1, whose catalytic mechanism had been researched clearly, participates in the covalent coupling of phycobilin and apoprotein in the form of chaperone, and its important amino acids have been confirmed. In order to identify the functional amino acid residues of CpcT1, chemical modification was conducted to arginine, histidine, tryptophan, lysine and amino acid carboxyl of CpcT1. The results indicated that the catalytic activity of the CpcT1 was changed. After the modification of arginine, tryptophan and histidine, site-directed mutations were performed to those highly conserved amino acids which were selected by means of homologous comparison. The mutated lyase, apoprotein and the enzymes that synthesize the phycobilins were recombined in Escherichia coli (E. coli) and in vitro, yielding chromoproteins, which were detected by fluorescence and UV absorption spectrometry. The spectra were compared with that of the chromoprotein catalyzed by wild type lyase CpcT1, achieving relative specific activities of the various mutants. Meanwhile, the mutants were expressed in E. coli, and then circular dichroism structure of near-UV region was determined. The results demonstrated that H33F, W175S, R97A, C137S and C116S influence the catalytic activity of CpcT1. Being different from wild CpcT1, a great deal of α helix was involved in the structure of circular dichroism of R97A and W13S. CpcT1 or its mutants and the enzymes that synthesize the phycobilins, were reconstituted in E. coli and detected by spectra to check the bounding of lyases and PCB. The results of spectra and SDS-PAGE confirm that CpcT1 and its mutants cannot bind phycobilin, differing from the catalytic mechanism of CpcS1.     

Cerato-ulmin—a wilting toxin of Dutch elm disease fungus

Cerato-ulmin, a toxin produced by Ceratocystis ulmi, the causal agent of Dutch elm disease, has been characterized as a small protein (128 residues) with a MW of ca 13000. The protein has a high content of cystine, proline, leucine, serine and aspartic acid/asparagine; it is low in histidine, lysine, arginine, isoleucine, phenylalanine and tyrosine and does not contain cysteine, methionine, or tryptophan. The amino acid sequence of the N-terminal region is: H₂N-Ala-Asp-Ser-Tyr-Asp-Pro-Cys-Thr-Gly-Leu-Leu-Gln-Lys-Ser-Pro-Gln-Cys-Cys-Asp-Thr-Asp-Ile-Leu-Gly-Val-Ser-Asp-Leu-Asp-Cys-. Toxic symptoms similar to those of Dutch elm disease can be elicited by cerato-ulmin in white elm shoot cuttings (Ulmus americana L.).     

Mechanisms and specificity of amino acid transport in Taenia crassiceps larvae (Cestoda)

The absorption of lysine, arginine, phenylalanine and methionine by Taenia crassiceps larvae is linear with respect to time for at least 2 min. Arginine uptake occurs by a mediated system and diffusion, and arginine, lysine and ornithine (in order of decreasing affinity) are completely competitive inhibitors of arginine uptake. The basic amino acid transport system has a higher affinity for l-amino acids than d-amino acids, and blocking the α-amino group of an amino acid destroys its inhibitory action. Phenylalanine uptake by T. crassiceps larvae is inhibited in a completely competitive fashion by serine, leucine, alanine, methionine, histidine, phenylalanine, tyrosine and tryptophan (in order of increasing affinity). Methionine apparently binds non-productively to the phenylalanine (aromatic amino acid-preferring) transport system. l-methionine uptake by larvae is inhibited more by d-alanine and d-valine than by their respective l-isomers, while d- and l-methionine inhibit l-methionine uptake equally well. The presence of an unsubstituted α-amino group is essential for an inhibitor to have a high affinity for the methionine transport system. Uptake of arginine, phenylalanine and methionine is Na⁺-insensitive, and both phenylalanine and methionine are accumulated by larvae against a concentration difference in the presence or absence of Na⁺. Arginine accumulation is precluded by its rapid metabolism to proline, ornithine and an unidentified compound.     

Interamino Acid Inhibition of Transport in Higher Plants : EVIDENCE FOR TWO TRANSPORT CHANNELS WITH ASCERTAINABLE AFFINITIES FOR AMINO ACIDS

Data from published experiments were analyzed to determine the number and specificities of amino acid transport channels in cells of higher plants. Each experiment measured the uptake of a labeled amino acid in the presence of unlabeled amino acids, used one at a time, in the incubating medium. The observed interamino acid inhibitions can be accounted for by two transport channels, each with characteristic affinities that were computed from the observed interamino acid inhibitions. The first channel is a general transport system with the following relative affinities for the amino acids: methionine 75, alanine 75, phenylalanine 64, tyrosine 64, leucine 63, cysteine 58, serine 57, glycine 56, tryptophan 54, glutamine 51, threonine 49, valine 44, isoleucine 44, glutamic acid 44, proline 43, histidine 33, lysine 32, asparagine 22, arginine 22, aspartic acid 18. The second channel is a basic amino acid tranport system with relative affinities for arginine, lysine, and histidine of 66, 39, and 21, respectively. The affinities for the other acids in the second channel are lower. Despite considerable diversity in the species, tissues, and solute concentrations employed in the experiments, multiple regression equations (Y = α + β₁X₁ + βX₂, in which Y is the observed transport inhibition and X₁ and X₂ are the relative transport affinities of the two channels) account for 50 to 99% of the variance in all but six experiments, five of which employed unusually high solute concentrations.     

Chemical modification and amino terminal sequence of calotropin DI from Calotropis gigantea

The effect of chemical modification of histidine, lysine, arginine, tryptophan and methionine residues on the enzymatic activity of calotropin DI has been studied. 1,3-Dibromoacetone inhibited the enzyme completely, indicating that a single histidine residue and a cysteine residue are involved in its catalytic activity. Its second bistidine residue was modified with diethyl pyrocarbonate without loss of activity. Modification of seven of its 13 lysine residues with 2,4,6-trinitrobenzene sulphonic acid led to 90% loss of its activity, but no single lysine residue appears to be essential for its activity. Four of the 12 arginine residues by 1,2-cyclohexanedione can be modified with little loss of activity. Modification of a single tryptophan residue and two methionine residues did not inhibit enzymatic activity. The blocked amino-terminal amino acid residue of calotropin DI has been identified as pyroglutamic acid. Its amino-terminal amino acid sequence to residue 14 has been determined and compared with that of papain. They show an extensive homology in their amino-terminal amino acid sequences.     

Influence of Forced-Feeding of Individual Essential Amino Acids on the Activities of All Urea Cycle Enzymes in Rat Liver

The response of all urea cycle enzymes, i.e. carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinase and arginase, has been determined in the liver of protein-depleted young rats which were forcibly fed individual essential l-amino acids along with or without caloric sources. The feeding of individual amino acids produced different effects on the level of each of the enzymes, and generally the response of carbamyl phosphate synthetase, argininosuccinate synthetase, argininosuccinase and arginase was greater than that of ornithine transcarbamylase. Of all the essential amino acids tested tryptophan was most effective on the elevation of these enzymes. Several amino acids, phenylalanine, leucine, threonine and methionine had also somewhat effect on the increase of some enzyme activities, but other amino acids had little or no effect on the response of these enzymes. On the contrary, histidine and lysine caused appreciable decrease of arginase activity. These enzyme activities in rats fed tryptophan alone were extremely higher than those of animals fed it along with caloric sources. The response level of the enzymes was essentially dependent on the tryptophan content in diets under the proper conditions. Tryptophan feeding did not produce any increase in both levels of urine and plasma urea despite the elevation of all urea cycle enzyme activities occured.     

Free amino acid concentrations in plasma of larval and adult hydropsychidae (Trichoptera)

Free amino acid concentrations in plasma of larval and adult Hydropsyche betteni and Cheumatopsyche pettiti were determined. Free amino acids in whole-body homogenates of these two species were also investigated.Larval haemoplasm of both species contained high concentrations of serine, proline, alanine, lysine, valine, threonine and arginine. Total free amino acid concentrations were consistently higher in C. pettiti larvae (33.1 mM) than in H. betteni larvae (24.6 mM).Haemoplasm from adult specimens of H. betteni contained high concentrations of serine and proline, followed by alanine, valine, lysine and histidine. Serine was found in very high concentrations in plasma from C. pettiti adults, followed by proline, glytamic acid, histidine and arginine. The mean total concentration of free amino acids was higher in C. pettiti adults (18.6 mM) than in H. betteni adults (13.3 mM).Comparison of free amino acids from haemoplasm and whole-body homogenates suggests that concentrations of free amino acids in body tissues differ from those present in plasma for these two insects.     

Effects of natural amino acids and sugars on activity of infusiorian cyclases

Natural amino acids and sugars in unicellular eukaryotes are known to regulate adenylyl cyclase (AC) and guanylyl cyclase (GC) systems that control the most important cell processes. The goal of the present work consisted in study of effects of natural amino acids and sugars and some of their derivatives on AC and GC activities of infusoria Tetrahymena pyriformis and Dileptus anser. Methionine, arginine, lysine, and tryptamine stimulated basic AC activity of T. pyriformis, whereas alanine, tyramine, and cysteine decreased it. Methionine, glycine, alanine, thyrosine, arginine, and to the lesser degree tryptamine and histidine stimulated AC of D. anser. The GC activity of T. pyriformis rose in the presence of tryptamine, tryptophane, histidine, arginine, and lysine, whereas glycine and aspartic acid, on the contrary, decreased it. Tryptamine, tryptophan, leucine, glutamic acid, serine, histidine, and alanine stimulated the GC activity of D. anser. Glucose, fructose, and sucrose stimulated the basal AC activity of both infusorians and GC of T. pyriformis, with glucose and sucrose increasing AC of T. pyriformis twice, while that of D. anser 4.5 times. Lactose stimulated AC and GC of T. pyriformis and was inefficient with respect to the D. anser cyclases, whereas mannose and galactose did not affect the enzyme activities in both infusorians. The study of the chemotactic response of the infusorians to amino acids and sugars indicates that involved in realization of this response can be signaling pathways both dependent on and independent of cyclic nucleotides. Thus, it has been established for the first time that several amino acids and sugars affect functional activity of enzymes with cyclase activity of the infusorians T. pyriformis and D. anser. This confirms the hypothesis that at early stages of evolution the large specter of comparatively simple natural molecules has a hormone-like action.     

A rapid procedure for detection of bacterial amino acid decarboxylases

The detection of decarboxylases of arginine, glutamic acid, histidine, lysine, ornithine, phenylalanine, tryptophan, and tyrosine in bacteria by thin-layer chromatography on polyamide sheets is described. The bacteria were grown on agar medium plates supplemented with eight amino acids at pH 5.5 for induction of amino acid decarboxylases, then transferred to amine-production media. The decarboxylation products in the spent media (amines and/or γ-amino-n-butyric acid) were dansylated and the dansyl derivatives were separated by thin-layer chromatography on polyamide sheets. This method requires only two separate incubations of the decarboxylase-induced bacteria in amine-production media for 1 h at 37°C for simultaneous detection of eight bacterial amino acid decarboxylases using 0.4 μl of the spent media.     

Role of the tissue free amino acids in adaptation of medicinal leeches <Emphasis Type="Italic">Hirudo medicinalis</Emphasis> L., 1758 to extreme climatic conditions

The first comparison of the spectra of free amino acids in tissues of the medicinal leeches H. medicinalis from different climatic and geographical Eurasian areas has been performed. Adaptation of H. medicinalis to extreme climatic conditions occurs via intensification of the amino acid metabolism resulting from a significant increase in the content of essential amino acids. Accumulation of arginine, histidine, and lysine (3.6-, 3.9-, and 2.0-fold increases, respectively) has proved to play a special protective role in adaptation of H. medicinalis to the low positive temperatures.     

Physiological and enzymatic aspects of histidine-mediated control of the tryptophan pathway

Summary It would thus appear that in Saccharomyces cerevisiae there are two forms of histidine-mediated control on the tryptophan pathway. In some strains histidine increases anthranilate synthetase and indole glycerol phosphate synthetase activities, while tryptophan synthetase decreases. In other strains histidine affects coordinately all enzymatic activities involved in tryptophan biosynthesis. The two groups of strains also differ in the formation, during the growth of the enzymatic activities involved in tryptophan biosynthesis. This difference in the relative rates at which the two enzymes are formed may explain the accumulation of intermediates in the cultural media of some strains. The derepression of anthranilate synthetase and indole glycerol phosphate synthetase activities by histidine is particularly manifest in the auxotrophic his₃ strains that show these activities very depressed in histidine starvation; large amounts of this amino acid stimulate them to a considerably greater extent than in prototrophic strains.Abbreviations IGP imidazole glycerol phosphate - InGP indole glycerol phosphate - ASase anthranilate synthetase - InGPase indole-3-glycerol phosphate synthetase - TSase tryptophan synthetase - Tris tris (hydroxymethyl)-aminomethane This investigation was supported by a research grant of C.N.R. (Consiglio Nazionale delle Ricerche, Roma).     

Understanding the Functional Roles of Amino Acid Residues in Enzyme Catalysis

The MACiE database contains 223 distinct step-wise enzyme reaction mechanisms and holds representatives from each EC sub-subclass where there is a crystal structure and sufficient evidence in the literature to support a mechanism. Each catalytic step of every reaction sequence in MACiE is fully annotated so that it includes the function of the catalytic residues involved in the reaction and the mechanism by which substrates are transformed into products. Using MACiE as a knowledge base, we have seen that the top 10 most catalytic residues are histidine, aspartate, glutamate, lysine, cysteine, arginine, serine, threonine, tyrosine and tryptophan. Of these only seven (cysteine, histidine, aspartate, lysine, serine, threonine and tyrosine) dominate catalysis and provide essentially five functional roles that are essential. Stabilisation is the most common and essential role for all classes of enzyme, followed by general acid/base (proton acceptor and proton donor) functionality, with nucleophilic addition following closely behind (nucleophile and nucleofuge). We investigated the occurrence of these residues in MACiE and the Catalytic Site Atlas and found that, as expected, certain residue types are associated with each functional role, with some residue types able to perform diverse roles. In addition, it was seen that different EC classes of enzyme have a tendency to employ different residues for catalysis. Further, we show that whilst the differences between EC classes in catalytic residue composition are not immediately obvious from the general classes of Ingold mechanisms, there is some weak correlation between the mechanisms involved in a given EC class and the functions that the catalytic amino acid residues are performing. The analysis presented here provides a valuable insight into the functional roles of catalytic amino acid residues, which may have applications in many aspects of enzymology, from the design of novel enzymes to the prediction and validation of enzyme reaction mechanisms.