Origin of amino acid homochirality: relationship with the RNA world and origin of tRNA aminoacylation

The origin of homochirality of l-amino acids has long been a mystery. Aminoacylation of tRNA might have provided chiral selectivity, since it is the first process encountered by amino acids and RNA. An RNA minihelix (progenitor of the modern tRNA) was aminoacylated by an aminoacyl phosphate oligonucleotide that exhibited a clear preference for l- as opposed to d-amino acids. A mirror-image RNA system with l-ribose exhibited the opposite selectivity, i.e., it exhibited an apparent preference for the d-amino acid. The selectivity for l-amino acids is based on the stereochemistry of RNA. The side chain of d-amino acids is located much closer to the terminal adenosine of the minihelix, causing them collide and interfere during the amino acid-transfer step. These results suggest that the putative RNA world that preceded the protein theatre determined the homochirality of l-amino acids through tRNA aminoacylation.     

Analysis of problems encountered in the determination of amino acid enantiomeric ratios by gas chromatography

A previously described procedure for determining the enantiomeric ratios of amino acids has produced inconsistent results when determining relatively low (≤0.110) d/l ratios. The method involves synthesis of diastereomeric N-trifluoroacetyl-l-prolyl-d/l-amino acid ester dipeptides which are resolved by gas chromatography (GC). We have found that triethylamine, which is added to maintain a basic pH during the coupling reaction, racemizes the chiral reagent N-trifluoroacetyl-l-prolyl chloride (TPC). Coupling of partially racemized TPC to d/l-amino acid esters results in the formation of four dipeptides (two pairs of enantiomers) instead of the expected two diastereomeric dipeptides. The enantiomeric dipeptides coelute on an achiral GC column, resulting in erroneous d/l ratios. More accurate d/l ratios are obtained by preparing the volatile N-trifluoroacetyl-d/l-amino acid isopropyl ester derivative which can be separated into its enantiomers on a chiral GC column such as the Chirasil-Val III (registered trademark of Applied Science Laboratories).     

Aromatic amino acid transaminase in rat intestine

The transamination of aromatic l-amino acids (5-hydroxytryptophan, tryptophan, tyrosine, phenylalanine and kynurenine) was shown to be catalysed by enzyme preparations from rat small intestine. On the basis of the partial purification and characterization of these aromatic amino acid transaminases, it is suggested that rat small intestine contains several kinds of aromatic amino acid transaminases.     

Reversal by aromatic amino acids of 2-thiazole-DL-alanine inhibition of Salmonella typhimurium

Of 21 l-amino acids tested (at 1.2 x 10(-4)m), only histidine and the aromatic amino acids (phenylalanine, tryptophan, and tyrosine) protect Salmonella typhimurium strains from inhibition of growth and immediately reverse the growth inhibition by 5 x 10(-4)m 2-thiazole-dl-alanine.     

Characterization of Bacillus thuringiensis L-isoleucine dioxygenase for production of useful amino acids

We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst.     

The amino acid transport systems of the autotrophically grown green alga Chlorella

The kinetic analysis of l-amino acid uptake by the green alga Chlorella revealed at least seven different uptake systems to be present in cells grown autotrophically with nitrate as nitrogen source. There is a ‘general system’ which transports most neutral and acidic amino acids, a system for short-chain neutral amino acids including proline, a system for basic amino acids including histidine, a special system for acidic amino acids, and specific systems for methionine, glutamine and threonine. The ‘general system’ is possibly the same as that which can be stimulated by incubation of cells in glucose plus ammonium (Sauer, N. (1984) Planta 161, 425–431). The incubation of Chlorella in glucose induces the increased synthesis of six amino acid uptake systems, namely the above-mentioned system for short-chain neutral amino acids, a threonine system, a methionine system, and a glutamine system. These results indicate that the uptake of l-amino acids by the green alga Chlorella is as complex as in other free-living organisms such as bacteria or yeast. The small number of amino acid uptake systems found in cells of higher plants, i.e. two or three, seems therefore to be a consequence of integration of the cells in a tissue supplying a relatively constant environment, and not a consequence of autotrophic growth on mineral carbon and mineral nitrogen.     

Enzymatic synthesis of amino acid-sugar alcohol conjugates in organic media

Amino acid-sugar alcohol conjugates were synthesized by a commercial serine protease, Optimase M-440, in organic media. Optimase M-440 showed broad substrate specificity towards N-t-Boc-protected l-amino acids as acyl donors and sugar alcohols as nucleophiles. Among various solvents tested Optimase M-440 showed the highest activity in pyridine. The regioselective acylation of the primary –OH groups of sugar alcohols gave the amino acid conjugates in good yields without byproducts.     

Cytotoxicity and inhibition of platelet aggregation caused by an l-amino acid oxidase from Bothrops leucurus venom

Background

Multifunctional l-amino acid oxidases (LAAOs) occur widely in snake venoms.

Methods

The l-AAO from Bothrops leucurus (Bl-LAAO) venom was purified using a combination of molecular exclusion and ion-exchange chromatographies. We report some biochemical features of Bl-LAAO associated with its effect on platelet function and its cytotoxicity.

Results

Bl-LAAO is a 60 kDa monomeric glycoprotein. Its N-terminal sequence shows high homology to other members of the snake-venom LAAO family. Bl-LAAO catalyzes oxidative deamination of l-amino acids with the generation of H₂O₂. The best substrates were: l-Met, l-Norleu, l-Leu, l-Phe and l-Trp. The effects of snake venom LAAOs in hemostasis, especially their action on platelet function remain largely unknown. Bl-LAAO dose-dependently inhibited platelet aggregation of both human PRP and washed platelets. Moreover, the purified enzyme exhibited a killing effect in vitro against Leishmania sp., promastigotes, with a very low EC₅₀ of 0.07 μM. Furthermore, the cytotoxicity of Bl-LAAO was observed in the stomach cancer MKN-45, adeno carcinoma HUTU, colorectal RKO and human fibroblast LL-24 cell lines. The enzyme released enough H₂O₂ in culture medium to induce apoptosis in cells in a dose- and time-dependent manner. The biological effects were inhibited by catalase.

Conclusion

Bl-LAAO, a major component of B. leucurus venom, is a cytotoxin acting primarily via the generation of high amounts of H₂O₂ which kill the cells.

General significance

These results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism.     

Effects of acetate and other short-chain fatty acids on sugar and amino acid uptake of Bacillus subtilis

Acetate and other short chain n-fatty acids (C(1)-C(6)) inhibit strongly the uptake of l-serine or other l-amino acids but inhibit only weakly that of alpha-methylglucoside or fructose, whether measured in whole cells of Bacillus subtilis or in membrane vesicles that have been energized with reduced nicotinamide adenine dinucleotide (NADH), l-alpha-glycerol phosphate, or ascorbate plus phenazine methosulfate. The acetate inhibition is noncompetitive, as was shown for l-alpha-aminoisobutyric acid uptake by whole cells and for l-serine uptake by membrane vesicles. In membrane preparations, neither NADH oxidation nor the reduction of cytochromes by NADH are affected by fatty acids. All of these effects are similar to those of 2, 4-dinitrophenol. It is concluded that the fatty acids "uncouple" the amino acid carrier proteins from the cytochrome-linked electron transport system (to which they may be coupled via protein interaction or via a cation gradient).     

Amino acid transport in a polyaromatic amino acid auxotroph of Saccharomyces cerevisiae

The initiation of growth of a polyaromatic auxotrophic mutant of Saccharomyces cerevisiae was inhibited by several amino acids, whereas growth of the parent prototroph was unaffected. A comparative investigation of amino acid transport in the two strains employing (14)C-labeled amino acids revealed that the transport of amino acids in S. cerevisiae was mediated by a general transport system responsible for the uptake of all neutral as well as basic amino acids. Both auxotrophic and prototrophic strains exhibited stereospecificity for l-amino acids and a K(m) ranging from 1.5 x 10(-5) to 5.0 x 10(-5) M. Optimal transport activity occurred at pH 5.7. Cycloheximide had no effect on amino acid uptake, indicating that protein synthesis was not a direct requirement for amino acid transport. Regulation of amino acid transport was subject to the concentration of amino acids in the free amino acid pool. Amino acid inhibition of the uptake of the aromatic amino acids by the aromatic auxotroph did not correlate directly with the effect of amino acids on the initiation of growth of the auxotroph but provides a partial explanation of this effect.     

ywfE in Bacillus subtilis codes for a novel enzyme, L-amino acid ligase

The ATP-dependent carboxylate-amine/thiol ligase superfamily is known to contain enzymes catalyzing the formation of various types of peptide, such as d-alanyl-d-alanine, polyglutamate, and gamma-peptide, but, curiously, no enzyme synthesizing alpha-dipeptides of l-amino acids is known. We attempted to find such an enzyme. By in silico screening based on the consensus sequence of the superfamily followed by an in vitro assay with purified enzyme to avoid the degradation of the peptide(s) synthesized, ywfE of Bacillus subtilis was found to code for the activity forming l-alanyl-l-glutamine from l-alanine and l-glutamine with hydrolysis of ATP to ADP. No AMP was formed, supporting the idea that the enzyme belongs to the superfamily. Surprisingly, the enzyme accepted a wide variety of l-amino acids. Among 231 combinations of l-amino acids tested, reaction products were obtained for 111 combinations and 44 kinds of alpha-dipeptides were confirmed by high-performance liquid chromatography analyses, while no tripeptide or longer peptide was detected and the d-amino acids were inert. From these results, we propose that ywfE encodes a new member of the superfamily, l-amino acid ligase.     

The consequence of sequence alteration of an amphipathic alpha-helical antimicrobial peptide and its diastereomers

The search for antibiotics with a new mode of action led to numerous studies on antibacterial peptides. Most of the studies were carried out with l-amino acid peptides possessing amphipathic alpha-helix or beta-sheet structures, which are known to be important for biological activities. Here we compared the effect of significantly altering the sequence of an amphipathic alpha-helical peptide (15 amino acids long) and its diastereomer (composed of both l- and d-amino acids) regarding their structure, function, and interaction with model membranes and intact bacteria. Interestingly, the effect of sequence alteration on biological function was similar for the l-amino acid peptides and the diastereomers, despite some differences in their structure in the membrane as revealed by attenuated total reflectance Fourier-transform infrared spectroscopy. However, whereas the all l-amino acid peptides were highly hemolytic, had low solubility, lost their activity in serum, and were fully cleaved by trypsin and proteinase K, the diastereomers were nonhemolytic and maintained full activity in serum. Furthermore, sequence alteration allowed making the diastereomers either fully, partially, or totally protected from degradation by the enzymes. Transmembrane potential depolarization experiments in model membranes and intact bacteria indicate that although the killing mechanism of the diastereomers is via membrane perturbation, it is also dependent on their ability to diffuse into the inner bacterial membrane. These data demonstrate the advantage of the diastereomers over their all l-amino acid counterparts as candidates for developing a repertoire of new target antibiotics with a potential for systemic use.     

Design and synthesis of vidarabine prodrugs as antiviral agents

5′-O-d- and l-amino acid derivatives and 5′-O-(d- and l-amino acid methyl ester phosphoramidate) derivatives of vidarabine (ara-A) were synthesized as vidarabine prodrugs. Some compounds were equi- or more potent in vitro than vidarabine against two pox viruses and their uptake by cultured cells was improved compared to the parent drug.     

An electrochemical sensor based on cellulose nanocrystal for the enantioselective discrimination of chiral amino acids

A novel electrochemical sensor based on 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose nanocrystals (TOCNCs) and l-cystines (l-Cys) modified Au electrode (TOCNC/l-Cys/Au) has been fabricated for detection and discrimination of the enantiomers of phenylalanine (Phe), leucine (Leu), and valine (Val). The three amino acids are in connection with metabolism diseases. The TOCNC/l-Cys/Au electrode exhibited obvious peak current difference for the amino acid enantiomers by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The TOCNCs on the electrode surface expressed different interactions with d- and l-amino acids, so the electrochemical recognitions of the three amino acid enantiomers were achieved. TOCNCs were characterized by Fourier transform infrared (FT-IR) and scanning electron microscopy (SEM). The modified electrodes were characterized by SEM and electrochemical techniques. According to DPV, peak currents of the two enantiomers decreased linearly with their concentrations. Furthermore, satisfactory results were obtained when this electrode was applied to measure the d- and l-Phe mixture. The experimental results show that TOCNCs are suitable material for chiral sensor. The contrast of serum sample of healthy people and patients with type 2 diabetes also was proposed, and significant difference was exhibited on the modified electrode. This work is significant for the screening, diagnosis, and treatment of multiple metabolic diseases.     

L-amino acids regulate parathyroid hormone secretion

Parathyroid hormone (PTH) secretion is acutely regulated by the extracellular Ca(2+)-sensing receptor (CaR). Thus, Ca(2+) ions, and to a lesser extent Mg(2+) ions, have been viewed as the principal physiological regulators of PTH secretion. Herein we show that in physiological concentrations, l-amino acids acutely and reversibly activated the extracellular Ca(2+)-sensing receptor in normal human parathyroid cells and inhibited parathyroid hormone secretion. Individual l-amino acids, especially of the aromatic and aliphatic classes, as well as plasma-like amino acid mixtures, stereoselectively mobilized Ca(2+) ions in normal human parathyroid cells in the presence but not the absence of the CaR agonists, extracellular Ca(2+) (Ca(2+)(o)), or spermine. The order of potency was l-Trp = l-Phe > l-His > l-Ala > l-Glu > l-Arg = l-Leu. CaR-active amino acids also acutely and reversibly suppressed PTH secretion at physiological ionized Ca(2+) concentrations. At a Ca(2+)(o) of 1.1 mm and an amino acid concentration of 1 mm, CaR-active amino acids (l-Phe = l-Trp > l-His = l-Ala), but not CaR-inactive amino acids (l-Leu and l-Arg), stereoselectively suppressed PTH secretion by up to 40%, similar to the effect of raising Ca(2+)(o) to 1.2 mm. A physiologically relevant increase in the -fold concentration of the plasma-like amino acid mixture (from 1x to 2x) also reversibly suppressed PTH secretion in the Ca(2+)(o) concentration range 1.05-1.25 mm. In conclusion, l-amino acids acutely and reversibly activate endogenous CaRs and suppress PTH secretion at physiological concentrations. The results indicate that l-amino acids are physiological regulators of PTH secretion and thus whole body calcium metabolism.     

Construction and characterization of Pichia pastoris strains for labeling aromatic amino acids in recombinant proteins

Strains of the methylotrophic yeast Pichia pastoris auxotrophic for the aromatic amino acids (tyrosine, phenylalanine, and tryptophan) have been constructed by targeted gene disruption for protein labeling applications. Three strains, with defects in ARO1 (coding for a homolog of the arom pentafunctional enzyme), ARO7 (coding for chorismate mutase), and TYR1 (coding for prephenate dehydrogenase), have been engineered in a P. pastoris ura3Delta1 parent strain using standard methods. The nutritional requirements of these auxotrophic strains have been characterized and their utility as expression hosts for labeling recombinant proteins has been demonstrated. All three strains show a surprising sensitivity to rich culture medium and must be grown in supplemented minimal medium. The tyr1::URA3 strain in particular is strongly inhibited by tryptophan, and to a lesser extent by phenylalanine, leucine, and isoleucine. Highly efficient incorporation of exogenously supplied amino acids by these three auxotroph strains has been demonstrated using recombinant galactose oxidase. Stereochemically pure l-amino acids and racemic d,l-mixtures serve nearly equally well to support protein expression and labeling. These strains allow efficient labeling of aromatic amino acids in recombinant proteins, supporting NMR structural biology and a wide range of other biophysical studies.     

Complete amino acid analysis of peptides and proteins after hydrolysis by a mixture of Sepharose-bound peptidases

Incubation with a mixture of Sepharose-bound peptidases was shown to result in the quantitative release of amino acids from certain peptides and S-aminoethylated proteins. Subtraction of the low background values of amino acids generated by the enzymes enables amino acid ratios of corticotrophin-(1-24)-tetracosapeptide to be determined with a standard deviation on repeat digestions of 3-5%. Good values were obtained for amino acids that are completely or partially destroyed on acid hydrolysis, i.e. tryptophan, tyrosine, serine, asparagine and glutamine. Experiments with peptides containing d-amino acids showed that the enzyme mixture is stereospecific and could therefore be used to detect the presence of d-residues in peptides. The enzyme mixture completely hydrolyses peptide fragments obtained after Edman degradation and should therefore be useful for determining sequences of peptides containing acid-labile amino acid residues. The activities of the bound enzymes were unaltered over a period of 7 months and they provide a simple, reproducible procedure for the quantitative determination of amino acids in peptides and proteins containing l-amino acids.     

Improved screening of microcystin genes and toxins in blue-green algal dietary supplements with PCR and a surface plasmon resonance biosensor

Microcystins (MCs) comprise a group of cyclic heptapeptide toxins that share a common backbone and have two variable l-amino acids that yield at least 21 known analogs of varying potency. These hepatotoxins and potential tumor promoters are produced by certain cyanobacteria, including Microcystis aeruginosa. The cyanobacterium M. aeruginosa blooms in freshwater lakes and can potentially co-occur with other species such as Aphanizomenon flos-aquae, which is targeted and harvested for the production of dietary supplements known as blue-green algae (BGA). BGA supplements are currently marketed in the U.S. and internationally as a product that may elevate mood, increase energy, and alleviate attention deficit hyperactivity disorder. However, the potential for BGA dietary supplements to be contaminated with MCs is of concern, and there are currently no validated methods for detection of MCs in these products. This research focused on establishing screening methods for toxic Microcystis and MCs in BGA supplements. A DNA-based method employing polymerase chain reaction (PCR) was used as a prescreening tool to evaluate the dietary supplements and to detect the presence of toxin genes (i.e., presence of toxic Microcystis). A rapid, sensitive surface plasmon resonance (SPR) biosensor, directed towards recognition of all MC forms, was also developed and validated. This improved SPR biosensor incorporates a commercial Adda-group antibody (Ab) that has the capacity for broader recognition of MCs than previously developed sensors for BGA supplements that rely solely on an arginine-reactive Ab and can quantitate MC levels down to 0.24 ng/mL (equivalent to 0.24 μg per gram of BGA supplement) in less than 10 min. Such a rapid, quantitative screening method may allow for further surveillance of BGA products to assist risk assessment efforts, establishment of regulatory guidance levels, and response to potential consumer complaints related to BGA products. The PCR technique and SPR biosensor may be used in concert as prescreening and screening tools, respectively or individually, thereby limiting the number of samples that must be evaluated with confirmatory methods.     

Some properties of Photosystem II preparations from the cyanobacterium Synechococcus sp. — presence of an l-amino acid oxidase in Photosystem II complexes from Synechococcus sp.

A highly active O₂-evolving Photosystem II complex has been purified from the cyanobacterium Synechococcus sp., and this complex has been compared with the Photosystem II complex previously isolated from this cyanobacterium (Ohno, T., Satoh, K. and Katoh, S. (1986) Biochim. Biophys. Acta 852, 1–8). Further treatment of the O₂-evolving complex with the detergent sodium taurodesoxycholate resulted in a complex which consisted mainly of the 47 and 40 kDa peptides and which had lost the O₂-evolving activity, but which could still reduce 2,6-dichlorophenolindophenol with 1,5-diphenylcarbazide. Previously, we have shown that a flavoprotein of 49 kDa which has an l-amino acid oxidase activity under certain conditions, is a component of highly active Photosystem II preparations from the cyanobacterium Anacystis nidulans (Pistorius, E.K. and Gau, A.E. (1986) FEBS Lett. 206, 243–248). Based on immunological studies with the antiserum raised against the l-amino acid oxidase protein from A. nidulans, we show that a protein which cross-reacts with this antiserum is present in the highly purified Photosystem II preparations from Synechococcus sp. Moreover, an l-amino acid oxidase activity could also be detected in Photosystem II preparations from Synechococcus sp. The enzyme preferentially oxidizes basic l-amino acids as l-arginine, l-ornithine, 2,3-diamino propionic acid and l-citrulline. In contrast to the enzyme from A. nidulansl-lysine is not oxidized. The here shown presence of an l-amino acid oxidase protein in Photosystem II preparations from Synechococcus sp. is an additional support of our hypothesis that a flavoprotein is a functional component of the water-oxidizing enzyme complex.