Amino acid substrate specificity of asparaginyl-,aspartyl- and glutaminyl-tRNA synthetase isolated from higher plants

AspNH₂-, Asp- and GluNH₂-tRNA synthetases were purified from Phaseolus aureus; their optimum assay conditions, substrate specificities and salt sensitivities were investigated. AspNH₂-tRNA synthetase from β-cyanoalanine-producing (Vicia sativa), and non-producing (P. aureus and V. faba) species was able to utilize the analogue as a substrate irrespective of the source of the enzyme. Asp-tRNA synthetase from P. aureus was able to utilize α-aminomalonate and threo-β-hydroxy Asp as a substrate. The transfer of ¹⁴C-GluNH₂ to tRNA, catalyzed by GluNH₂-tRNA synthetase, was only inhibited by high concentrations of those analogues tested; albizziine was the most efficient, but no difference could be demonstrated between the substrate specificities of the enzyme isolated from an albizziine-producer (A. julibrissin and a non-producer (P. aureus) species.     

Neutral amino acid transport in Leishmania promastigotes

Neutral amino acid transport was investigated in Leishmania promastigotes. Proline and alanine transport occur against their concentration gradient although there is a very rapid (40% at 30 min) conversion of proline to alanine. Uptake of these amino acids occurs by a sodium-independent route which is completely eliminated by addition of CCCP or KCN. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for proline and alanine are 80 μM and 63 μM with $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ values of 6.4 and 7.2 nmol/min per mg dry weight, respectively. Countertransport of proline, alanine and phenylalanine was measured by loading the cells with a variety of neutral amino acids and proline analogs, followed by CCCP addition. The effect of aminooxyacetic acid, an inhibitor of alanine aminotransferase (EC 2.6.1.2), on proline and alanine countertransport was also examined. The results obtained are consistent with the presence of at least two systems for neutral amino acid transport in Leishmania promastigotes.     

Tyrosine O-glucoside and dopamine 3-O-glucoside in seeds of Entada pursaetha

l-Tyrosine O-glucoside (I) and dopamine-3-O-glucoside (II) have been isolated from seeds of Entada pursaetha DC. The structures have been established by spectroscopic methods, identification of hydrolysis products and comparison with synthetic material. Syntheses are described of II, dopamine 4-glucoside and tyramine-O-glucoside.     

2S,4R-carboxy-2-acetylamino-4-piperidine dans les feuilles de Calliandra haematocephala

A new naturally occurring pipecolic acid derivative has been isolated from leaves of the legume Calliandra haematocephala. Its structure was shown to be 2S,4R-carboxy-2-acetylamino-4-piperidine by chemical and spectrosopic methods.     

Use of β3-methionine as an amino acid substrate of Escherichia coli methionyl-tRNA synthetase

Polypeptides containing β-amino acids are attractive tools for the design of novel proteins having unique properties of medical or industrial interest. Incorporation of β-amino acids in vivo requires the development of efficient aminoacyl-tRNA synthetases specific of these non-canonical amino acids. Here, we have performed a detailed structural and biochemical study of the recognition and use of β³-Met by Escherichia coli methionyl-tRNA synthetase (MetRS). We show that MetRS binds β³-Met with a 24-fold lower affinity but catalyzes the esterification of the non-canonical amino acid onto tRNA with a rate lowered by three orders of magnitude. Accurate measurements of the catalytic parameters required careful consideration of the presence of contaminating α-Met in β³-Met commercial samples. The 1.45 Å crystal structure of the MetRS: β³-Met complex shows that β³-Met binds the enzyme essentially like α-Met, but the carboxylate moiety is mobile and not adequately positioned to react with ATP for aminoacyl adenylate formation. This study provides structural and biochemical bases for engineering MetRS with improved β³-Met aminoacylation capabilities.     

Simple and efficient synthesis of chiral amino alcohols with an amino acid-based skeleton

Sodium bis(2-methoxyethoxy)aluminum hydride, NaAlH₂(OCH₂CH₂OCH₃)₂, commercially known as Vitride® or Red-Al®, enables rapid synthesis of pure optically active N-protected amino alcohols and peptide alcohols in very high yields. The method is very simple and attractive, as it does not require an additional step of N-protected amino acid derivatization and proceeds without the loss of enantiomeric homogeneity.     

The activation of amino acid analogues by phenylalanyl- and tyrosyl-tRNA synthetases from plants

Partially purified preparations of Phe- and Tyr-tRNA synthetases were obtained from seed or seedlings of Phaseolus aureus, Delonix regia and Caesalpinia tinctoria, and the ability of a variety of structural analogues of Phe or Tyr to act as alternative substrates or inhibitors was tested. 3-Hydroxymethylphenylalanine, a natural product of C. tinctoria, formed a particularly effective substrate for the Tyr-tRNA synthetase from P. aureus. The structural features commensurate with substrate activity in an analogue molecule are discussed.     

2(S),4(R)-4-(β-d-Galactopyranosyloxy)-4-isobutyl-glutamic acid: A new amino acid in Reseda odorata

2(S),4(R)-4-(β-d-Galactopyranosyloxy)-4-isobutylglutamic acid (I) has been isolated from the flowers of Reseda odorata, wherein it occurs in substantial quantity. Hydrolysis of I gives d-galactose, 2(S),4(R)-4-hydroxy-4-isobutylglutamic acid (II) and 3(R),5(S)-3-hydroxy-3-isobutyl-2-pyrrolidone-5-carboxylic acid (III) and its treatment with nitrous acid yields a galactoside of a non-nitrogenous hydroxy acid lactone (IV). The structures of I and its degradation products are supported by PMR, ¹³C-NMR and other spectroscopic methods. ¹³C-NMR spectroscopy of the model compound 2-(β-d-galactopyranosyloxy)isobutyric acid confirmed the structure of the natural product. The S- (or l-) configuration at C(2) in the amino acid moiety of I has been established by the use of the Clough—Lutz—Jirgenson rule and the R-configuration at C(4) of the same unit has been assigned tentatively. I represents the first example of a glycoside of a higher plant amino acid in which the carbohydrate residue is linked to an aliphatic hydroxy group.     

X-ray studies on crystalline complexes involving amino acids and peptides. Part XIV: Closed conformation and head-to-tail arrangement in a new crystal form of L-histidine L-aspartate monohydrate

A new form of L-histidine L-aspartate monohydrate crystallizes in space group P2₂ witha = 5.131(1),b = 6.881(1),c= 18.277(2) Å,β= 97.26(1)° and Z = 2. The structure has been solved by the direct methods and refined to anR value of 0.044 for 1377 observed reflections. Both the amino acid molecules in the complex assume the energetically least favourable allowed conformation with the side chains staggered between the α-amino and α-scarboxylate groups. This results in characteristic distortions in some bond angles. The unlike molecules aggregate into alternating double layers with water molecules sandwiched between the two layers in the aspartate double layer. The molecules in each layer are arranged in a head-to-tail fashion. The aggregation pattern in the complex is fundamentally similar to that in other binary complexes involving commonly occurring L amino acids, although the molecules aggregate into single layers in them. The distribution of crystallographic (and local) symmetry elements in the old form of the complex is very different from that in the new form. So is the conformation of half the histidine molecules. Yet, the basic features of molecular aggregation, particularly the nature and the orientation of head-to-tail sequences, remain the same in both the forms. This supports the thesis that the characteristic aggregation patterns observed in crystal structures represent an intrinsic property of amino acid aggregation.     

ds-erythro-2-amino-3,4-dihydroxybutanoic acid,a constituent in the edible mushroom,lyophyllum ulmarium

An unusual amino acid occurring in the fruiting bodies of Lyophyllum ulmarium was identified as d_s-erythro- 2-amino-3,4-dihydroxybutanoic acid, which is a compound found for the first time in biological materials.     

Distribution of α-amino-β-methylaminopropionic acid in Cycas

α-Amino-β-methylaminopropionic acid, previously isolated from seeds of Cycas circinalis, has now been identified either free or bound in all th     

Effects of glucocorticoids on the gene expression of nutrient transporters in different rabbit intestinal segments

Glucocorticoids (GCs) are counterregulatory hormones with broad effects on the digestion and absorption of dietary carbohydrates, lipids and proteins, but the underlying molecular mechanisms of these effects remain unclear. The present experiment was conducted to investigate the main expression sites of nutrient transporters and the effects of GCs on the gene expression of these transporters in the rabbit small intestine. The results showed that peptide transporter 1 (PepT1), facultative amino acid transporter (rBAT), neutral amino acid transporter (B⁰AT), excitatory amino acid transporter 3 (EAAT3), sodium-glucose transporter 1 (SGLT1) and glucose transporter 5 (GLUT5) were mainly expressed in the distal segment, glucose transporter 2 (GLUT2) and fatty-acid-binding protein 4 (FATP4) were mainly expressed in the proximal segment and cationic amino acid transporter 1 (CAT1) was mainly expressed in the middle segment of the rabbit small intestine. In addition, we analysed the effects of 3 h (short-term) or 7 days (long-term) dexamethasone (DEX) treatment on the gene expression of most nutrient transporters. The results showed that short-term DEX treatment significantly decreased PepT1, B⁰AT, EAAT3, rBAT and SGLT1 expressions in all small intestinal segments, while it significantly decreased GLUT2 in the duodenum and FATP4 in the duodenum and ileum (P < 0.05). Long-term DEX treatment also significantly decreased PepT1, CAT1, B⁰AT, EAAT3, rBAT and SGLT1 in all small intestinal segments and significantly decreased GLUT2 in the jejunum and FATP4 in the ileum (P < 0.05). In conclusion, DEX could decrease the gene expression of most nutrient transporters (except GLUT5) and affect the transport of intestinal amino acids, monosaccharides and fatty acids.     

NTS2-selective neurotensin mimetics with tetrahydrofuran amino acids

Stimulation of the NTS2 neurotensin receptor causes antipsychotic effects and leads to a promotion of the μ-opioid-independent antinociception, which is important in the modulation of tonic pain sensitivity. We report the synthesis and properties of a small library of peptidic agonists based on the active neurotensin fragment NT(8–13). Two tetrahydrofuran amino acid derivatives were synthesized to replace Tyr¹¹ in NT(8–13). Additionally, Arg⁸, Arg⁹, and Ile¹² of the lead peptide were exchanged by Lys, Lys, and Gly, respectively. The new compounds showed substantial NTS2 binding affinity and up to 1000-fold selectivity over NTS1. The highest selectivity (K_i(NTS2): 29 nM, K_i(NTS1): 35,000 nM) was observed for the peptide analog 17R^trans.     

l-2-Oxalylamino-3-aminopropionic acid,an isomer of Lathyrus sativus neurotoxin

The first chemical synthesis of l-2-oxalylamino-3-aminopropionic acid, an isomer of the Lathyrus sativus neurotoxin, is described. Studies on its biological properties are reported. Experiments with l-3-[¹⁴C-oxalyl]amino-2- aminopropionic acid show that the amount of 2-oxalylamino isomer detectable in seed extracts can be accounted for by rearrangement which occurs during isolation.     

Erythro-γ-hydroxyhomo-L-arginine: An amino acid from seed of Lonchocarpus costaricensis, and its preferential interaction with borate

A new non-protein amino acid, erythro-γ-hydroxyhomo-L-arginine has been isolated from seed of Lonchocarpus costaricensis by exploiting its property of interacting with borate ions. For structural comparisons, threo-γ-hydroxyhomo-L-arginine was isolated from seed of Lathyrus tingitanus and erythro-γ-hydroxyarginine from Vicia unijuga by novel procedures. The reasons for the interaction of borate with the erythro- but not the threo-forms of these amino acids are discussed.     

Design and synthesis of glucose-templated proline-lysine chimera: polyfunctional amino acid chimera with high prolyl cis amide rotamer population

We describe the synthesis of two glucose-templated proline-lysine chimeras (GlcTProLysCs) that differ in the stereochemistry of the hydroxymethyl substituent at the C-5′ position of the pyrrolidine ring. The key synthetic steps involve C-glycosylation of an exocyclic glucose-based epoxide with allyltributylstannane, which affords functionalized C-ketosides containing an α-hydroxy ester moiety; introduction of an amino group at C-2 through stereoselective reductive amination; and regioselective installation of the azide group at C-6 on the glucose scaffold. Incorporation of these chimeras into the model peptides Ac-GlcTProLysC-NHMe and Ac-GlcTProLysC-OMe demonstrates that the stereochemistry of the hydroxymethyl substituent at the C-5′ position has a profound effect on the equilibrium constant of prolyl amide cis/trans isomerization. The equilibrium constant K_c/t for the peptide mimic Ac-GlcTProLysC-NHMe with C-5′(R) stereochemistry was determined to be 3.03 ± 0.04, while the K_t/c for the C-5′(S) diastereoisomer was 0.56 ± 0.04 in D₂O. Temperature coefficient experiments indicate that the origin of these effects is derived from two critical hydrogen bonds involving the C-5′ hydroxymethyl substituent: one to the N-terminal amide carbonyl group, and the other to the primary amino group in the glucose moiety.     

Genetic screening of Lactobacillus sakei and Lactobacillus curvatus strains for their peptidolytic system and amino acid metabolism, and comparison of their volatilomes in a model system

A total of 51 Lactobacillus sakei and 28 Lactobacillus curvatus strains from different origins were screened for their potential to produce biogenic amines (BAs), and for their diversity of peptidolytic systems and specific aminotransferases (AraT, BcaT) that initiate amino acid conversion to volatiles relevant for aroma formation in meat products. The profiles of volatiles formed (volatilomes) were analysed in the headspace of fermentations by solid phase microextraction followed by GC-MS analysis. Tyramine-forming potential was detected only within L. curvatus and was strain-dependent. Histamine decarboxylase (HDC) activity could only be detected in one L. sakei strain, previously described as histidine decarboxylase positive (HDC⁺). Peptide transporters and peptidases were nearly ubiquitous in L. sakei and only a few strains lacked single peptidases. In L. curvatus, differences were detected in the occurrence of peptidase genes detected with PCR primers derived from L. sakei. All strains lacked known aminotransferases specific for branched-chain amino acids (BCAAs) and aromatic amino acids (ACAAs). Although L. sakei is suggested as a genetically very heterogenous species, and relatedness between L. curvatus and L. sakei at the genomic level is rather low, they appeared to be nearly uniform in the genes forming the peptidolytic system. The volatilomes of L. sakei and L. curvatus strains were qualitatively nearly identical. However, slight differences in the formation of single volatile compounds and the interaction with staphylococci may impact upon sausage fermentation which occurs over a period of many weeks. Among the compounds expected to contribute to the aroma were dimethyldisulphide, 3-methyl-1-butanol, acetic acid, 1-butanol and butanoic acid.     

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.).     

Ds-erythro-2-amino-4-ethoxy-3-hydroxybutanoic acid from the fruiting bodies of the edible mushroom,Lyophyllum ulmarium

A new β-hydroxy amino acid isolated from the fruiting bodies of Lyophyllum ulmarium was identified as D_s,-erythro-2-amino-4-ethoxy-3-hydroxybutanoic acid by chemical degradation and spectroscopic analyses.