Characterization of an L-α,ß-diaminopropionic acid polymer with comb-like structure isolated from a poly(ε-L-lysine)-producing Streptomyces sp.

Polymers of basic amino acids function as polycationic compounds under physiological conditions and exhibit intriguing biological properties, such as antimicrobial and antiviral activities, immunopotentiating ability, and DNA-binding activity. Poly(ε-L-lysine) (ε-PL) produced by some strains of Streptomyces spp. is a cationic homopolymer of L-lysine linking between ε-amino and α-carboxylic acid functional groups and has been used as a food preservative based on its biocompatibility and biodegradability. An ε-PL-producing strain of Streptomyces sp. USE-33 was found to secrete a novel polycationic substance into its culture broth along with ε-PL. High-performance liquid chromatography analyses and one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) experiments, accompanied by NMR titration studies, revealed that the secreted substance was poly[ß-(L-diaminopropionyl-L-diaminopropionic acid)], PAP, characterized by an isopeptide backbone linking between the ß-amino and α-carboxylic acid groups of L-α,ß-diaminopropionic acid (L-Dpr) with pendent L-Dpr residues. PAP had a molecular weight of 500 to 1400, and copolymers composed of the two amino acids L-Dpr and L-lysine were not detected in the producer strain USE-33. The strain coproduced high levels of the two poly(amino acid)s in the presence of glycerol, citrate, and ammonium sulfate at pH 4.0 in a two-stage cultivation procedure. PAP exhibited strong inhibitory activities against several yeasts and weaker activities against bacteria than ε-PL. PAP may share a number of biological functions with ε-PL, and the use of PAP along with ε-PL has potential as a specific and advanced material for technical applications in various fields.Key points• Novel cationic poly(amino acid) was found in an ε-PL-producing Streptomyces species.• The l-α,ß-diaminopropionic acid polymer was characterized by a comb-like structure.• The novel poly(amino acid), PAP, exhibited antibacterial and antifungal activities.

The Stereocontrolled Biosynthesis of Mirror-Symmetric 2,4-Diaminobutyric Acid Homopolymers Is Critically Governed by Adenylation Activations.

Among the four bioactive cationic homo-poly(amino acids) discovered in nature, two are mirror-image isomers of poly(2,4-diaminobutyric acid) (poly-Dab) whose biosynthesis has long been unexplained. Their structural analogy plausibly suggested that they could share a common biosynthetic pathway utilizing ε-poly(l-lysine) synthetase-like enzymology but with an unprecedented process for enantiomeric inversion of polymer building blocks. To investigate this possibility, we comparatively explored the biosynthesis of poly-l-Dab and its mirror-image isomer poly-d-Dab in Streptomyces celluloflavus USE31 and Streptoalloteichus hindustanus NBRC15115, respectively, through genome mining, genetic inactivation, and heterologous expression combined with biochemical assays. While they shared the same biosynthetic pathway, the poly-d-Dab biosynthetic gene cluster additionally harbored the racemase gene. The critical finding that poly-d-Dab synthetase, in contrast to the synthetase generating the l-isomer, selectively activated d-Dab through adenylation conclusively demonstrated that free diffusible d-Dab preactivationally generated by the racemase is directly activated to be incorporated into the polymer. Our study thus represents the first demonstration of the stereoselective biosynthesis of a nonribosomal peptide governed by adenylation activity for a d-amino acid other than alanine. In silico sequence comparison between poly-Dab synthetases allowed us to identify amino acid residues potentially responsible for the discrimination of Dab enantiomers. Our results will provide significant insight not only for the future discovery of novel bioactive cationic poly(amino acids) but also for the creation of designer nonribosomal peptides with d-configuration.

Isolation and spectral characterization of thermally generated multi-Z-isomers of lycopene and the theoretically preferred pathway to di-Z-isomers.

Lycopene has a large number of geometric isomers caused by E/Z isomerization at arbitrary sites within the 11 conjugated double bonds, offering varying characteristics related to features such as antioxidant capacity and bioavailability. However, the geometric structures of only a few lycopene Z-isomers have been thoroughly identified from natural sources. In this study, seven multi-Z-isomers of lycopene, (9Z,13'Z)-, (5Z,13Z,9'Z)-, (9Z,9'Z)-, (5Z,13'Z)-, (5Z,9'Z)-, (5Z,9Z,5'Z)-, and (5Z,9Z)-lycopene, were obtained from tomato samples by thermal isomerization, and then isolated by elaborate chromatography, and fully assigned using proton nuclear magnetic resonance. Moreover, the theoretically preferred pathway from (all-E)-lycopene to di-Z-isomers was examined with a computational approach using a Gaussian program. Fine-tuning of the HPLC separation conditions led to the discovery of novel multi-Z-isomers, and whose formation was supported by advanced theoretical calculations.

Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase.

BACKGROUND: SERCA maintains intracellular Ca2+ homeostasis by sequestering cytosolic Ca2+ into SR/ER stores. Two primary fatty acid amides (PFAAs), oleamide (18:19-cis) and linoleamide (18:29,12-cis), induce an increase in intracellular Ca2+ levels, which might be caused by their inhibition of SERCA. METHODS: Three major SERCA isoforms, rSERCA1a, hSERCA2b, and hSERCA3a, were individually overexpressed in COS-1 cells, and the inhibitory action of PFAAs on Ca2+-ATPase activity of SERCA was examined. RESULTS: The Ca2+-ATPase activity of each SERCA was inhibited in a concentration-dependent manner strongly by linoleamide (IC50 15-53µM) and partially by oleamide (IC50 8.3-34µM). Inhibition by other PFAAs, such as stearamide (18:0) and elaidamide (18:19-trans), was hardly or slightly observed. With increasing dose, linoleamide decreased the apparent affinity for Ca2+ and the apparent maximum velocity of Ca2+-ATPase activity of all SERCAs tested. Oleamide also lowered these values for hSERCA3a. Meanwhile, oleamide uniquely reduced the apparent Ca2+ affinity of rSERCA1a and hSERCA2b: the reduction was considerably attenuated above certain concentrations of oleamide. The dissociation constants for SERCA interaction varied from 6 to 45µM in linoleamide and from 1.6 to 55µM in oleamide depending on the isoform. CONCLUSIONS: Linoleamide and oleamide inhibit SERCA activity in the micromolar concentration range, and in a different manner. Both amides mainly suppress SERCA activity by lowering the Ca2+ affinity of the enzyme. GENERAL SIGNIFICANCE: Our findings imply a novel role of these PFAAs as modulators of intracellular Ca2+ homeostasis via regulation of SERCA activity.

Identification of novel inhibitors of human SPCA2.

To identify specific inhibitors of the human secretary pathway Ca(2+)-ATPase 2 (hSPCA2), a recombinant hSPCA2 was expressed in Saccharomyces cerevisiae, and purified by Co(2+)-chelating chromatography. The isolated hSPCA2 catalyzed ATP hydrolysis in the presence of Ca(2+) ions. The Ca(2+) dissociation constant for ATPase activation was 25 nM. hSPCA2 activity was inhibited by thapsigargin, 2,2'-methylenebis(6-tert-butyl-p-cresol), and 4-octylphenol in the low-micromolar concentration range. Unexpectedly, the organic solvent wash from standard laboratory polypropylene microtubes showed strong inhibitory potency toward hSPCA2 activity. The extract was found to comprise mainly primary fatty acid amides (PFAAs) by NMR analysis. Individual PFAAs, especially oleamide and linoleamide, almost completely inhibited hSPCA2 activity with IC50 values of 7.5 µM and 3.8 µM, respectively.

Isolation and characterization of (15Z)-lycopene thermally generated from a natural source.

(15Z)-Lycopene was prepared by thermal isomerization of (all-E)-lycopene derived from tomatoes, and isolated by using a series of chromatographies. The fine red crystalline powder of (15Z)-lycopene was obtained from 556 mg of (all-E)-lycopene with a yield of 0.6 mg (purity: reversed-phase HPLC, 97.2%; normal-phase HPLC, ≥99.9%), and (1)H and (13)C NMR spectra of the isomer were fully assigned. More refined computational analyses that considered differences in the energy levels of the conformers involved in isomerization have also determined the stabilities of (15Z)-lycopene and other geometric isomers, along with the activation energies during isomerization from the all-E form. The fine control of conditions for HPLC separation and an advanced theoretical insight into geometric isomerization have led to the discovery of the 15Z-isomer generated from a natural source.

Crystal structure of 1,4-dieth-oxy-9,10-anthra-quinone.

The asymmetric unit of the title compound, C18H16O4, contains two crystallographically independent mol-ecules. The anthra-quinone ring systems are slightly bent with dihedral angles of 2.33 (8) and 13.31 (9)° between the two terminal benzene rings. In the crystal, the two independent mol-ecules adopt slipped-parallel π-overlap with an average inter-planar distance of 3.45 Å, forming a dimer; the centroid-centroid distances of the π-π inter-actions are 3.6659 (15)-3.8987 (15) Å. The mol-ecules are also linked by C-H⋯O inter-actions, forming a tape structure along the a-axis direction. The crystal packing is characterized by a dimer-herringbone pattern.

Enhanced E/Z Isomerization of (All-E)-lycopene by Employing Iron(III) Chloride as a Catalyst.

UNLABELLED: Catalytic isomerization of (all-E)-lycopene to Z-isomers using iron(III) chloride was investigated and optimized under various conditions of solvents, concentrations of iron(III) chloride, and reaction temperatures. The total contents of Z-isomers converted were higher in the order of CH2 Cl2 (78.4%) > benzene (61.4%) > acetone (51.5%) > ethyl acetate (50.8%) at 20 °C for 3 h using 1.0 × 10(-3) mg/mL iron(III) chloride for 0.1 mg/mL (all-E)-lycopene. However, the decomposition of lycopene was markedly accelerated in CH2 Cl2 : the remaining lycopene after the reaction for 3 h and 12 h was only 79.4% and 47.5%, respectively. As the concentration of catalyst increased in acetone, the Z-isomerization ratio of lycopene increased to more than 80%, followed by rapid degradation of lycopene to undetectable levels using >4.0 × 10(-3) mg/mL iron(III) chloride with the above concentration of (all-E)-lycopene. Finally, greater isomerization (79.9%) was attained at 60 °C in acetone for 3 h in the presence of 1.0 × 10(-3) mg/mL iron(III) chloride, largely without decomposition of lycopene (remaining ratio of total amount of lycopene isomers after the reaction, 96.5%). As iron(III) chloride has found general use as a food additive for iron fortification and acetone is also widely used in the food field, this method can be applied to the food and beverage processing industry. PRACTICAL APPLICATION: The dietary intake of lycopene, a natural red pigment found in brightly colored vegetables and fruits such as tomatoes and watermelons, has been reported to lower the risk of some diseases, including cancer. Lycopene molecules occur naturally in a long and "straight" shape, but on the other hand lycopene molecules with "bent" forms are highly absorbed by living cells, and showed good antioxidant activity. This study has demonstrated the efficient production of the "bent" lycopene using ionic iron as an accelerator, which is often contained in nutritional supplements.

Spectral characterisation of Z-isomers of lycopene formed during heat treatment and solvent effects on the E/Z isomerisation process.

The geometric isomerisation of (all-E)-lycopene, purified from tomato paste, was investigated in various organic solvents. Isomerisation ratios to the Z-isomers of lycopene in CH2Cl2 and CHCl3 over 24h were calculated to be 19.7% and 11.4% at 4°C and 77.8% and 48.4% at 50°C, respectively. In CH2Br2, more than 60% was attained in the first several hours, independent of temperature. The predominant Z-isomers obtained thermally, (9Z)-lycopene and (13Z)-lycopene, were purified and their absorption maxima and molar extinction coefficients in hexane were determined for the first time. Absorption values at 460 nm were also measured for both Z-isomers along with (all-E)-lycopene to accurately evaluate their concentrations by HPLC analysis. This approach successfully revealed that (13Z)-lycopene formed predominantly in benzene or CHCl3 at 50°C; in contrast, the 5Z-isomer was preferentially obtained in CH2Cl2 or CH2Br2.

Crystal structure of 2-bromo-1,4-dihy-droxy-9,10-anthra-quinone.

In an attempt to brominate 1,4-diprop-oxy-9,10-anthra-quinone, a mixture of products, including the title compound, C14H7BrO4, was obtained. The mol-ecule is essentially planar (r.m.s. deviation = 0.029 Å) and two intra-molecular O-H⋯O hydrogen bonds occur. In the crystal, the mol-ecules are linked by weak C-H⋯O hydrogen bonds, Br⋯O contacts [3.240 (5) Å], and π-π stacking inter-actions [shortest centroid-centroid separation = 3.562 (4) Å], generating a three-dimensional network.

Photosensitized E/Z isomerization of (all-E)-lycopene aiming at practical applications.

Photoisomerization of (all-E)-lycopene to the corresponding Z-isomers was investigated under visible to middle-infrared light irradiation in the presence of several sensitizers, including edible ones. Highly purified (all-E)-lycopene from tomato paste was isomerized to Z-isomers to the extent of 46.4-57.4% after irradiation with the sensitizers for 60 min in acetone, in which a thermodynamically stable isomer of (5Z)-lycopene was predominantly generated, whereas kinetically preferable (9Z)- and (13Z)-lycopenes were dominant without sensitizer. Examination of the time course of photoisomerization demonstrated that the highest isomerization efficiency (80.4%) was attained using erythrosine as the sensitizer under 480-600 nm light irradiation in hexane for 60 min, a protocol that successfully suppressed the degradation of lycopene. (5Z)-Lycopene, reported as a more bioavailable isomer, was again predominantly produced with erythrosine and rose bengal in each solvent.

Characterization and thermal isomerization of (all-E)-lycopene.

A large amount of (all-E)-lycopene was successfully purified from tomato paste using an improved method that included a procedure to wash crystalline powder with acetone. The total yield of the pure (all-E) form was at least 30%. The melting point of (all-E)-lycopene was determined to be 176.35 °C by differential scanning calorimetry (DSC) measurements. Bathochromic shifts were observed in the absorption maxima of all solvents tested (at most a 36 nm shift for λ2 in carbon disulfide, as was observed in hexane) and were accompanied by absorbance decreases, namely, a hypochromic effect, showing a higher correlation between the position and the intensity of the main absorption bands. This bathochromic shift was dependent upon the polarizability of the solvent rather than its polarity. The structure of (all-E)-lycopene in CDCl3 and C6D6 was identified on the basis of one- and two-dimensional nuclear magnetic resonance (NMR) spectra, including (1)H and (13)C NMR, homonuclear correlation spectroscopy ((1)H-(1)H COSY), heteronuclear multiple-quantum coherence (HMQC), and heteronuclear multiple-bond connectivity (HMBC). The rate constants of the decrease in (all-E)-lycopene with hexane and benzene were calculated to be 3.19 × 10(-5) and 3.55 × 10(-5) s(-1), respectively. The equilibrium constants between (all-E) and (13Z) isomers were estimated to be 0.29 in hexane and 0.31 in benzene, respectively, from the point at which the amount of (13Z)-lycopene reached its maximum.

A novel alkaline esterase from Sporosarcina sp. nov. strain eSP04 catalyzing the hydrolysis of a wide variety of aryl-carboxylic acid esters.

A novel esterase showing activity specific for esters of aryl-carboxylic acids was discovered in Sporosarcina sp. nov., which was identified by the 16S rDNA sequencing method in addition to morphological and physiological analyses. The aryl-carboxylesterase (named EstAC) was purified 780-fold from crude cell extracts by a 5-step procedure. EstAC was characterized as a monomeric protein with a molecular weight of 43,000, an optimum pH of around 9.0, and an optimum temperature of 40 °C. The pH optimum and the effects of inhibitors together with an internal amino acid sequence suggested that EstAC is a member of family VIII esterases. EstAC was found to be highly active on a wide variety of substrates such as alkyl benzoates, alkyl phenylacetates, ethyl α- or ß-substituted phenylpropionates, dialkyl terephthalates, dimethyl isophthalate, and ethylene glycol dibenzoate. However, monomethyl terephthalate was not hydrolyzed. It was suggested that EstAC had 4-hydroxybenzoyl and cinnamoyl esterase activities as well.

High-yield production of short chain length poly(epsilon-L-lysine) consisting of 5-20 residues by Streptomyces aureofaciens, and its antimicrobial activity.

Poly(epsilon-L-lysine) (epsilon-PL) is a naturally-occurring L-lysine homopolymer having antimicrobial activity. A newly-isolated strain of Streptomyces aureofaciens produced a short chain length epsilon-PL consisting of 5-20 residues at the highest production level of 4.5 g l(-1). This epsilon-PL had different spectra in terms of antimicrobial activity from the epsilon-PL that is now used as a food preservative. The high productivity was based on multiple metabolic pathways for L-lysine synthesis, and a great flux from L-lysine to epsilon-PL. The usefulness of this new epsilon-PL and its producing strain was discussed.

Poly(gamma-L-diaminobutanoic acid), a novel poly(amino acid), coproduced with poly(epsilon-L-lysine) by two strains of Streptomyces celluloflavus.

Two poly(epsilon-L-lysine) (epsilon-PL) producer strains of Streptomyces celluloflavus secreted a novel polymeric substance into their culture broths along with epsilon-PL. Three types of HPLC analysis plus one- and two-dimensional 1H and 13C nuclear magnetic resonance experiments revealed that the secreted substance was poly(gamma-L-diaminobutanoic acid) (gamma-PAB), an L-alpha,gamma-diaminobutanoic acid (L-DAB) homopolymer linking between y-amino and alpha-carboxylic acid functional groups. The gamma-PABs from the two strains had an identical chemical structure, and the same number-average molecular weight of 2100-2200. No copolymers composed of the two amino acids L-DAB and L-lysine were found in either of the broths from the producers. Both strains coproduced high levels of the two poly(amino acid)s in the presence of SO4(2-) at pH 4.0 and 4.5 L min(-1) aeration in a 5-L jar fermentor. gamma-PAB exhibited strong inhibitory activities against various yeasts and weaker actions against bacteria than epsilon-PL. gamma-PAB may have various biological functions similar to epsilon-PL, and the use of gamma-PAB along with epsilon-PL would be advantageous for technical applications in various fields.

Biosynthesis of nearly monodispersed poly(epsilon-L-lysine) in Streptomyces species.

Poly(epsilon-L-lysine) (epsilon-PL) is a naturally occurring poly(amino acid) characterized by a unique structure linking epsilon-amino and carboxyl groups of L-lysine. Due to its various functions and its biodegradability and non-toxicity, the epsilon-PL polymer has attracted increasing attention in recent years. epsilon-PL is frequently found in various strains of Streptomyces sp. This review gives an up-to-date overview regarding the biosynthesis of epsilon-PL focussing mainly on results obtained from ten newly isolated producer strains, using the two-stage culture method of cell growth and epsilon-PL production cultures. The production of nearly monodispersed epsilon-PL is covered together with the development of epsilon-PL specific hydrolases and the release of synthesized epsilon-PL into the culture broth. From these results, coupled with the termination of polymerization through nucleophilic chain transfer, the biosynthetic mechanism of the polymer is discussed.

Substantially monodispersed poly(epsilon-L-lysine)s frequently occurred in newly isolated strains of Streptomyces sp.

The presence of poly(epsilon-L-lysine) (epsilon-PL) was found quite frequently by screening various strains of Streptomyces sp. Most of the ten newly obtained epsilon-PLs, when they were produced from glucose, showed a polydispersity index of Mw/Mn = 1.01 using ion-pair chromatography analysis. The polymers were classified into five groups according to their chain lengths. The average numbers of residues in the five groups were 32, 28, 25, 19, and 16, respectively. The use of glycerol instead of glucose resulted in decreases of 10 to 20% in the Mn and slight increases in the Mw/Mn. These observations indicated the chain length and polydispersity of epsilon-PL were primarily determined by each producer strain. Proton and 13C NMR analysis revealed the signals of glycerol-derived ester at the C terminus of the polymer from several producers including the first discovered S. albulus strain, although the percentages of the ester were low under our culture conditions. These results, coupled with the previous observation that SO4(2-) was essential for the polymer production, led to discussion on the mechanistic aspects of monomer activation, elongation, and termination in the biosynthesis of epsilon-PL.

Biosynthesis of poly(epsilon-L-lysine)s in two newly isolated strains of Streptomyces sp.

The biosynthesis of poly(epsilon-L-lysine) (epsilon-PL) in the two newly isolated strains of Streptomyces lydicus USE-11 (USE-11) and Streptomyces sp. USE-51 (USE-51) was studied by a newly developed two-stage culture method of cell growth at pH 6.8 and epsilon-PL production at pH 4.5. USE-11 synthesized epsilon-PL consisting of about 28 residues at a high production level, whereas USE-51 did the polymer with 15 ones at a low level. The secreted epsilon-PLs in culture media were digested in a neutral pH range with a peptide hydrolase(s) produced by the epsilon-PL producers. The optimum production levels were presumed to be dependent upon the inherent epsilon-PL synthesis machinery of each producer. The production in USE-51 was sharply dependent upon cell density as was often observed in the production of antibiotics, whereas that in USE-11 was scarcely affected by the density. The SO(4)(2-) was found to be essential for the epsilon-PL production in both strains. This might suggest the involvement of a thiol group in the polymerization reactions including the activation of L-lysine. This study indicates that USE-11 is a most suitable strain for the exploration of the epsilon-PL biosynthesis at the molecular level as well as for the technical applications.