Configuration Of Amino Acids Research Articles

Pullularins E and F, Two New Peptides from the Endophytic Fungus Bionectria ochroleuca Isolated from the Mangrove Plant Sonneratia caseolaris

Chemical investigation of the EtOAc extract of the endophytic fungus Bionectria ochroleuca, isolated from the inner leaf tissues of the plant Sonneratia caseolaris (Sonneratiaceae) from Hainan island (China), yielded two new peptides, pullularins E and F (1 and 2) together with three known compounds (3–5). The structures of the new compounds were unambiguously determined on the basis of one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of amino acids were determined by HPLC analysis of acid hydrolysates using Marfey’s method. The isolated compounds exhibited pronounced to moderate cytotoxic activity against the mouse lymphoma cells (L5178Y) with EC50 values ranging between 0.1 and 6.7 µg/mL.

Two piperazic acid-containing cyclic hexapeptides from Streptomyces alboflavus 313

Two novel cyclic hexapeptides, designated NW-G10 (1) and NW-G11 (2), were isolated from the fermentation broth of Streptomyces alboflavus 313. Their relative structures were elucidated on the basis of extensive spectroscopic analysis, and the absolute configurations of several constituent amino acids were determined by Marfey's method. NW-G10 (1) and NW-G11 (2) exhibited significant activity against Gram-positive bacteria, such as Bacillus cereus, Bacillus subtilis and Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA), but they are not active against gram negatives.

Chemical‐Biological Exploration of the Limits of the Ras De‐ and Repalmitoylating Machinery

A dynamic de-/repalmitoylation cycle determines localization and activity of H- and N-Ras. This combined cellular de- and repalmitoylation machinery has been shown to be substrate tolerant--it accepts variation of amino acid sequence, structure and configuration. Here, semisynthetic Ras-proteins in which the C-terminal amino acids are replaced by peptoid residues are used to reveal the first limitations of substrate recognition by the de- and repalmitoylating machinery.

Insights into the molecular mechanism of tolerance to carboxylic acid amide (CAA) fungicides in Pythium aphanidermatum

Tolerance to the oomycete-specific carboxylic acid amide (CAA) fungicides is a poorly understood mechanism in Pythium species. The root-rot and damping-off causative agent Pythium aphanidermatum and the CAA fungicide mandipropamid (MPD) were used to investigate the molecular basis of CAA tolerance. Five genes putatively involved in carbohydrate synthesis were identified and characterised: one chitin synthase gene, PaChs, and four cellulose synthase genes PaCesA1 to PaCesA4, of which PaCesA3 encodes the MPD target enzyme. These genes were differentially expressed throughout the life cycle of P. aphanidermatum. Mycelium treated with MPD concentrations slightly affecting mycelial growth did not cause a change in PaCesA3 expression nor a strong upregulation of PaCesA homologues. The high tolerance level of P. aphanidermatum and the lack of PaCesA upregulation imply that MPD tolerance is the result of a specific amino acid configuration in the cellulose synthase 3 (CesA3) target enzyme. Indeed, P. aphanidermatum displays the amino acid L1109 which is also associated with MPD resistance in artificial mutants of Phytophthora species. It is concluded that MPD tolerance in P. aphanidermatum is not caused by compensatory mechanisms but most likely by an inherent target-site configuration in PaCesA3 that hinders MPD binding to the enzyme pocket.

The cellulose synthase 3 (CesA3) gene of oomycetes: structure, phylogeny and influence on sensitivity to carboxylic acid amide (CAA) fungicides

Proper disease control is very important to minimize yield losses caused by oomycetes in many crops. Today, oomycete control is partially achieved by breeding for resistance, but mainly by application of single-site mode of action fungicides including the carboxylic acid amides (CAAs). Despite having mostly specific targets, fungicidal activity can differ even in species belonging to the same phylum but the underlying mechanisms are often poorly understood.In an attempt to elucidate the phylogenetic basis and underlying molecular mechanism of sensitivity and tolerance to CAAs, the cellulose synthase 3 (CesA3) gene was isolated and characterized, encoding the target site of this fungicide class. The CesA3 gene was present in all 25 species included in this study representing the orders Albuginales, Leptomitales, Peronosporales, Pythiales, Rhipidiales and Saprolegniales, and based on phylogenetic analyses, enabled good resolution of all the different taxonomic orders. Sensitivity assays using the CAA fungicide mandipropamid (MPD) demonstrated that only species belonging to the Peronosporales were inhibited by the fungicide. Molecular data provided evidence, that the observed difference in sensitivity to CAAs between Peronosporales and CAA tolerant species is most likely caused by an inherent amino acid configuration at position 1109 in CesA3 possibly affecting fungicide binding. The present study not only succeeded in linking CAA sensitivity of various oomycetes to the inherent CesA3 target site configuration, but could also relate it to the broader phylogenetic context.

JBIR-78 and JBIR-95: Phenylacetylated Peptides Isolated from Kibdelosporangium sp. AK-AA56

The search for metabolites of Kibdelosporangium sp. AK-AA56 resulted in the discovery of novel N-phenylacetylated peptides, JBIR-78 (1) and JBIR-95 (2). Compounds 1 and 2 were established to be N-phenylacetylated heptapeptides by extensive NMR and HRESIMS analyses. The absolute configuration of the standard amino acids including a cysteic acid moiety was determined using Marfey's method on the acid hydrolysates of 1 and 2. The relative and absolute configurations of a nonstandard amino acid, β-hydroxyleucine, were elucidated using the J-based and modified Mosher's methods, respectively. In an antimicrobial test, 1 showed antibacterial activity against Micrococcus luteus.

Searching for new cell-penetrating agents: hybrid cyclobutane–proline γ,γ-peptides

Two generations of hybrid γ,γ-peptides containing cyclobutane amino acids and cis-γ-amino-L-proline joined in alternation have been synthesized and their capacity to cross the eukaryotic cell membrane has been evaluated. The first generation consists of di-, tetra- and hexapeptides, and their properties have been analyzed as well as the influence of peptide length and chirality of the cyclobutane residues. Results have shown that the absolute configuration of the cyclobutane amino acid does not have a relevant influence. The second generation consists of hybrid γ,γ-hexapeptides with a common backbone and distinct side chains introduced with different linkage types through the α-amino group (N(α)) of the proline monomers. These peptides have been shown to be non-toxic towards HeLa cells and to internalize them effectively, the best results being obtained for the peptides with a spacer of five carbons between the N(α) atom and the guanidinium group. The introduction of cyclobutane residues inside the sequence affords a good balance between charge and hydrophobicity, reducing the number of positive charges. This results in lower toxicity and similar cell-uptake properties when compared to previously described peptide agents.

Prediction of DNA-binding proteins from relational features

BackgroundThe process of protein-DNA binding has an essential role in the biological processing of genetic information. We use relational machine learning to predict DNA-binding propensity of proteins from their structures. Automatically discovered structural features are able to capture some characteristic spatial configurations of amino acids in proteins.ResultsPrediction based only on structural relational features already achieves competitive results to existing methods based on physicochemical properties on several protein datasets. Predictive performance is further improved when structural features are combined with physicochemical features. Moreover, the structural features provide some insights not revealed by physicochemical features. Our method is able to detect common spatial substructures. We demonstrate this in experiments with zinc finger proteins.ConclusionsWe introduced a novel approach for DNA-binding propensity prediction using relational machine learning which could potentially be used also for protein function prediction in general.

Fijimycins A–C, three antibacterial etamycin-class depsipeptides from a marine-derived Streptomyces sp.

Three new depsipeptides, fijimycins A–C (1–3), together with the known etamycin A (4), were isolated and identified from the fermentation broth of strain CNS-575, a Streptomyces sp. cultured from a marine sediment sample collected off Nasese, Fiji. The planar structures of the new fijimycins were assigned by combined interpretation of NMR and MS/MS spectroscopic data. These assignments were complicated by the fact that 1–3 occurred as complex amide conformational mixtures. The absolute configurations of the component amino acids were established using the Marfey’s method. Fijimycins A–C, and etamycin A, were shown to possess significant in vitro antibacterial activity against three methicillin-resistant Staphylococcus aureus (MRSA) strains with MIC100 values between 4 and 16μgmL−1.

New C14-surfactin methyl ester from the marine bacterium Bacillus pumilus KMM 456

New C14-surfactin methyl ester and eight earlier described surfactins were isolated from the marine bacterium Bacillus pumilus associated with zoantharia Palythoa sp. The structure of the compounds isolated was established based on the data from amino acid analysis, one- and two-dimensional NMR spectroscopy, and tandem nanoelectrospray mass spectrometry. The absolute configurations of amino acids were determined by Marfey’s method. Cytotoxic activity of a number of surfactins with respect to the tumor cells HCT-116 and MDA-MB-231 was studied.

ChemInform Abstract: Elucidation of the Absolute Configurations of Amino Acids and Amines by the Modified Mosher′s Method.

Abstract Application of the modified Mosher's method to the N -MTPA derivatives of several amino acid esters and acyclic amines indicates that this method may be generally used to determine the absolute configurations of the α-carbons of amino compounds of less than 0.1 mg amount.

ChemInform Abstract: New Strategies for an Efficient Removal of the 9-Fluorenylmethoxycarbonyl (Fmoc) Protecting Group in the Peptide Synthesis.

The aluminium trichloride/toluene system is investigated as a novel, unusual and straightforward reagent for the removal of the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group in the solution peptide synthesis. This procedure avoids any undesired side reactions, such as the frequently observed inversion of the amino acid configuration.

Enantioselective CD analysis of amino acids based on chiral amplification with a stereodynamic probe

The condensation between stereolabile 1,8-bis(3′-formyl-4′-hydroxyphenyl)naphthalene, 1, and two amino acid molecules results in the formation of chiral diimines exhibiting strong CD signals. This reaction has been used to develop a chiroptical sensing method for the determination of the absolute configuration and enantiomeric composition of unprotected amino acids. This sensing approach is based on distinctive chiral amplification due to central-to-axial chirality induction within the diimine scaffold formed and does not require the use of an enantiopure ligand or metal complex.

Erythrochelin – a hydroxamate‐type siderophore predicted from the genome of Saccharopolyspora erythraea

The class of nonribosomally assembled siderophores encompasses a multitude of structurally diverse natural products. The genome of the erythromycin-producing strain Saccharopolyspora erythraea contains 25 secondary metabolite gene clusters that are mostly considered to be orphan, including two that are responsible for siderophore assembly. In the present study, we report the isolation and structural elucidation of the hydroxamate-type tetrapeptide siderophore erythrochelin, the first nonribosomal peptide synthetase-derived natural product of S. erythraea. In an attempt to substitute the traditional activity assay-guided isolation of novel secondary metabolites, we have employed a dedicated radio-LC-MS methodology to identify nonribosomal peptides of cryptic gene clusters in the industrially relevant strain. This methodology was based on transcriptome data and adenylation domain specificity prediction and resulted in the detection of a radiolabeled ornithine-inheriting hydroxamate-type siderophore. The improvement of siderophore production enabled the elucidation of the overall structure via NMR and MS(n) analysis and hydrolysate-derivatization for the determination of the amino acid configuration. The sequence of the tetrapeptide siderophore erythrochelin was determined to be D-alpha-N-acetyl-delta-N-acetyl-delta-N-hydroxyornithine-D-serine-cyclo(L-delta-N-hydroxyornithine-L-delta-N-acetyl-delta-N-hydroxyornithine). The results derived from the structural and functional characterization of erythrochelin enabled the proposal of a biosynthetic pathway. In this model, the tetrapeptide is assembled by the nonribosomal peptide synthetase EtcD, involving unusual initiation- and cyclorelease-mechanisms.

Hantupeptins B and C, cytotoxic cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula

Hantupeptins B (2) and C (3) were isolated, along with the previously reported hantupeptin A (1), from the marine cyanobacterium, Lyngbya majuscula, collected from Pulau Hantu Besar, Singapore. Their structures were elucidated by interpretation of extensive 1D and 2D NMR spectroscopic data. Compounds 2 and 3 are cyclic depsipeptides consisting of five alpha-amino/hydroxy acid residues, including phenyllactic acid, proline, N-methyl-valine, valine, N-methyl-isoleucine, and a beta-hydroxy acid unit with different degrees of unsaturation at the terminal end of each molecule. The absolute configurations of the common amino acids and phenyllactic acid were determined by the advanced Marfey's and chiral HPLC analyses, respectively. The complete stereochemistry of 3-hydroxy-2-methyl-7-octynoic acid moiety in hantupeptin A was elucidated by a combination of homonuclear J-resolved 2D NMR experiments and by Mosher's method. Hantupeptins B and C showed moderate in vitro cytotoxicity when tested against MOLT-4 (leukemic) and MCF-7 (breast cancer) cell lines.

Inversion of the Configuration of a Single Stereocenter in a β‐Heptapeptide Leads to Drastic Changes in its Interaction with Phospholipid Bilayers

One small change for a molecule…︁ The change in the configuration of a single amino acid in a β3-heptapeptide can lead to remarkable changes in the physicochemical properties of the peptide with respect to the interaction with lipid vesicles (liposomes) formed from DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine).

The Elucidation of the Structure of Thermotoga maritima Peptidoglycan Reveals Two Novel Types of Cross-link

Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of L- and D-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. The first monomer was the d-lysine-containing disaccharide-tripeptide in which the D-Glu-D-Lys bond had the unusual gamma-->epsilon arrangement (GlcNAc-MurNAc-L-Ala-gamma-D-Glu-epsilon-D-Lys). The second monomer was the conventional disaccharide-tetrapeptide (GlcNAc-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala). The first dimer contained a disaccharide-L-Ala as the acyl donor cross-linked to the alpha-amine of D-Lys in a tripeptide acceptor stem with the sequence of the first monomer. In the second dimer, donor and acceptor stems with the sequences of the second and first monomers, respectively, were connected by a D-Ala4-alpha-D-Lys3 cross-link. The cross-linking index was 10 with an average chain length of 30 disaccharide units. The structure of the peptidoglycan of T. maritima revealed for the first time the key role of D-Lys in peptidoglycan synthesis, both as a surrogate of L-Lys or meso-diaminopimelic acid at the third position of peptide stems and in the formation of novel cross-links of the L-Ala1(alpha-->alpha)D-Lys3 and D-Ala4(alpha-->alpha)D-Lys3 types.

Exploration of Mononucleotides as Fixed Ligands towards Chiral Discrimination of Hexose Monosaccharides by the Kinetic Method

The most recent version of the kinetic method, i.e. fixed ligand method, is applied towards chiral discrimination of three pairs of enantiomeric hexose monosaccharides under mass spectral conditions. Naturally occurring mononucleotides are used as fixed ligands (FL) and the amino acids are selected as the chiral references (ref) to discriminate the analyte (A), the enantiomers of glucose, mannose and galactose. Chiral discrimination is achieved by investigating the collision-induced dissociation spectra of trimeric complex ion, [Ni(II)(FL)(ref)(A)-H](+) generated by electrospraying the solution mixture of D- or L- analyte (A), FL, amino acid (ref) and NiCl(2). The relative abundance of product ions resulting from the competitive loss of reference amino acid and analyte are considered for measuring the degree of chiral discrimination by applying the kinetic method. L-Asp, L-Thr, L-Glu, L-Trp and L-Ser are found as suitable reference compounds. Among the tested mononucleotides (5'AMP, 5'GMP, 5'CMP, 5'UMP and 5'TMP), 5'GMP is found to be the best for the studied analytes. Chiral discrimination is found to depend on the nature of the monosaccharide, the functional groups present in the side chain of reference amino acids and the configuration of reference amino acids.

New Chiral Derivatizing Agents: Convenient Determination of Absolute Configurations of Free Amino Acids by 1H NMR

The chiral carbonate reagents 5 allow for the direct and unambiguous determination of the absolute configurations of a wide range of free amino acids using (1)H NMR. By using a approximately 3:1 mixture of (S)-5 and (R)-5, absolute configurations of the corresponding carbamates are determined by only analyzing the nitrogen protons.

Structurally based epitope analysis of major histocompatibility complex class I–related chain A (MICA) antibody specificity patterns

Recent studies have suggested a clinical significance to the detection of anti–major histocompatibility complex class I–related chain A (MICA) antibodies in transplantation. We have developed an eplet-based version of the HLAMatchmaker algorithm to assess the epitope specificity of these antibodies. Molecular viewing of the MICA structure and the determination of amino acid sequence differences between MICA alleles has yielded a repertoire of 38 potentially immunogenic MICA eplets. These eplets are based on the functional epitope structure that considers a configuration of amino acids within a 3-Ångstrom radius of an antibody-accessible polymorphic residue on the molecular surface. In this study MICA-reactive sera were screened in Luminex assays with single MICA allele panels and analyzed with HLAMatchmaker. We identified reactivity patterns that correspond to eplet-specific antibodies to identify of unacceptable MICA mismatches including those alleles that have not been tested with the serum. In conclusion, HLAMatchmaker is a useful algorithm to analyze the reactivity patterns of anti-MICA antibodies and the determination of MICA mismatch acceptability at the structural level.