Streptomyces metabolites in divergent microbial interactions.

Streptomyces and related bacteria produce a wide variety of secondary metabolites. Of these, many compounds have industrial applications, but the question of why this group of microorganism produces such various kinds of biologically active substances has not yet been clearly answered. Here, we overview the results from our studies on the novel function and role of Streptomyces metabolites.

Natural low-molecular mass organic compounds with oxidase activity as organocatalysts.

Organocatalysts, low-molecular mass organic compounds composed of nonmetallic elements, are often used in organic synthesis, but there have been no reports of organocatalysts of biological origin that function in vivo. Here, we report that actinorhodin (ACT), a natural product derived from Streptomyces coelicolor A3(2), acts as a biocatalyst. We purified ACT and assayed its catalytic activity in the oxidation of L-ascorbic acid and L-cysteine as substrates by analytical methods for enzymes.

Improvement of stability of nitrile hydratase via protein fragment swapping.

Nitrile hydratase (NHase), which catalyzes the hydration of nitriles to amides, is the key enzyme for the production of amides in industries. However, the poor stability of this enzyme under the reaction conditions is a drawback of its industrial application. In this study, we aimed to improve the stability of NHase (PpNHase) from Pseudomonas putida NRRL-18668 using a homologous protein fragment swapping strategy.

Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide.

Self-assembling amphipathic peptides (SAPs) are the peptides that can spontaneously assemble into ordered nanostructures. It has been reported that the attachment of SAPs to the N- or C-terminus of an enzyme can benefit the thermo-stability of the enzyme. Here, we discovered that the thermo-stability and product tolerance of nitrile hydratase (NHase) were enhanced by fusing with two of the SAPs (EAK16 and ELK16).

Development of mass spectrometry selected reaction monitoring method for quantitation and pharmacokinetic study of stepharine in rabbit plasma.

Highly sensitive liquid chromatography mass spectrometry method on triple quadrupole (QQQ) mass spectrometer was successfully applied for pharmacokinetic study of stepharine in rabbit plasma. Specific ion transitions of stepharine protonated precursor ion were selected and recorded in the certain retention time employing dynamic selected reaction monitoring mode. The developed method facilitated quantitative measurements of stepharine in plasma samples in linear range of five orders of magnitude with high accuracy and low standard deviation coefficient and pharmacokinetics parameters were calculated.

Dietary supplementation with Lactobacilli improves emergency granulopoiesis in protein-malnourished mice and enhances respiratory innate immune response.

This work studied the effect of protein malnutrition on the hemato-immune response to the respiratory challenge with Streptococcus pneumoniae and evaluated whether the dietary recovery with a probiotic strain has a beneficial effect in that response. Three important conclusions can be inferred from the results presented in this work: a) protein-malnutrition significantly impairs the emergency myelopoiesis induced by the generation of the innate immune response against pneumococcal infection; b) repletion of malnourished mice with treatments including nasally or orally administered Lactobacillus rhamnosus CRL1505 are able to significantly accelerate the recovery of granulopoiesis and improve innate immunity and; c) the immunological mechanisms involved in the protective effect of immunobiotics vary according to the route of administration. The study demonstrated that dietary recovery of malnourished mice with oral or nasal administration of L.

A new synthetic route to N-benzyl carboxamides through the reverse reaction of N-substituted formamide deformylase.

Previously, we isolated a new enzyme, N-substituted formamide deformylase, that catalyzes the hydrolysis of N-substituted formamide to the corresponding amine and formate (H. Fukatsu, Y. Hashimoto, M.

Enhanced oxidative activities of cytochrome P450Rhf from Rhodococcus sp. expressed using the hyper-inducible expression system.

Bacterial cytochrome P450 enzymes catalyze the oxidative biotransformation of various types of compounds. Although the functional expression of Actinomycetes P450 in a closely related heterologous host can serve as a useful biocatalyst in whole-cell biotransformation assays, the co-expression of an electron transfer partner is required. To overcome this limitation, P450Rhf from Rhodococcus sp.

Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis.

Aldoxime dehydratase (OxdA), which is a unique heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. Unlike the utilization of H(2)O(2) or O(2) as a mediator of catalysis by other heme-containing enzymes (e.g.

High expression levels of chitinase genes in Streptomyces coelicolor A3(2) grown in soil.

Although Streptomyces species are major chitin-degraders in soil ecosystems, the expression of the diverse chitinase genes within Streptomyces coelicolor grown in soil has not been assessed. As a first step, the induction pattern of nine chitinase genes in S. coelicolor growing in autoclaved soil was compared with those in liquid cultures.

Conductometric and light scattering studies on the complexation between cationic polyelectrolyte nanogel and anionic polyion.

This work aims to provide a basic understanding of the water dispersibility of a 1:1 stoichiometric polyelectrolyte complex (SPEC) in water in the absence of low-molecular-weight salts. We studied the complexation of a linear polyanion, potassium poly(vinyl alcohol sulfate) (KPVS), with a cationic polyelectrolyte nanogel (CPENG) composed of a lightly cross-linked copolymer of N-isopropylacrylamide and 1-vinylimidazole, in an aqueous salt-free solution (pH 3 and 25 °C), as a function of the molar mixing ratio (Mmr) of anionic to cationic groups. Also studied for comparison was the complexation of KPVS with poly(diallyldimethylammonium chloride) (PDDA), which is a standard reaction in colloid titration.

Unique biogenesis of high-molecular mass multimeric metalloenzyme nitrile hydratase: intermediates and a proposed mechanism for self-subunit swapping maturation.

Rhodococcus rhodochrous J1 produces high- and low-molecular mass nitrile hydratases (H-NHase and L-NHase, respectively), depending on the inducer. The incorporation of cobalt into L-NHase has been found to depend on the α-subunit exchange between cobalt-free L-NHase (apo-L-NHase) and its cobalt-containing mediator, NhlAE (holo-NhlAE), this novel mode of post-translational maturation having been named self-subunit swapping and NhlE having been recognized as a self-subunit swapping chaperone. We discovered an H-NHase maturation mediator, NhhAG, consisting of NhhG and the α-subunit of H-NHase.

Novel isonitrile hydratase involved in isonitrile metabolism.

We previously discovered N-substituted formamide deformylase (NfdA) in Arthrobacter pascens F164, which degrades N-substituted formamide (Fukatsu, H., Hashimoto, Y., Goda, M.

On the water dispersibility of a 1:1 stoichiometric complex between a cationic nanogel and linear polyanion.

This work aimed to obtain information on the water dispersibility of a 1:1 stoichiometric polyelectrolyte nanogel complex (SPENC). We synthesized a cationic polyelectrolyte nanogel (CPENG) composed of a cross-linked copolymer of 1-vinylimidazole and N-isopropylacrylamide. SPENC was then prepared at 25 degrees C from the mixing of equimolar amounts (based on fixed charges) of CPENG and potassium poly(vinyl alcohol) sulfate, which were dissolved in an aqueous solution without adding salt and at pH 3.

Self-subunit swapping chaperone needed for the maturation of multimeric metalloenzyme nitrile hydratase by a subunit exchange mechanism also carries out the oxidation of the metal ligand cysteine residues and insertion of cobalt.

The incorporation of cobalt into low molecular mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 has been found to depend on the alpha-subunit exchange between cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues) and its cobalt-containing mediator (holo-NhlAE containing Cys-SO(2)(-) and Cys-SO(-) metal ligands), this novel mode of post-translational maturation having been named self-subunit swapping, and NhlE having been recognized as a self-subunit swapping chaperone (Zhou, Z., Hashimoto, Y., Shiraki, K.

Mitocryptide-2: purification, identification, and characterization of a novel cryptide that activates neutrophils.

Neutrophils are a class of leukocytes involved in innate immunity by monitoring and scavenging invading microorganisms and toxic substances. The actions of neutrophils in damaged tissues are still not well understood, particularly in the early stage of inflammation, and as-yet-unknown neutrophil-activating substances are proposed to induce their acute transmigration and activation. Here, we isolated and identified from porcine hearts a neutrophil-activating peptide.

On the temperature-responsive polymers and gels based on N-propylacrylamides and N-propylmethacrylamides.

We studied the behavior in water of polymers, microgels, and macrogels based on the following four monomers: N-isopropylacrylamide (NiPA), N-isopropylmethacrylamide (NiPMA), N-n-propylacrylamide (NnPA), and N-n-propylmethacrylamide (NnPMA). The thermal phase separation of polymers in water as well as of microgels in the aqueous dispersion was examined by a combination of turbidity measurements and differential scanning calorimetry (DSC). The hydrodynamic radius of microgels and the swelling degree of macrogels (fine cylindrical bulk gels) were also examined as a function of temperature using the dynamic light scattering and the microscopic method, respectively.

Estrogenic and Acetylcholinesterase-Enhancement Activity of a New Isoflavone, 7,2',4'-Trihydroxyisoflavone-4'-O-beta-D-Glucopyranoside from Crotalaria Sessililflora.

A new isoflavone, 7,2',4'-trihydroxyisoflavone-4'-O-beta-D-glucopyranoside has been isolated from the aerial part of Crotalaria sessiliflora. The isoflavone glucoside enhanced the proliferation of the MCF-7 human breast cancer cell line, which possesses estrogen receptor (ER) and responds to estrogen in culture. The estrogenic property of the isoflavone glucoside was blocked by the known ER antagonist tamoxifen, indicating the involvement of the ER.

Elucidation of structural requirements of mastoparan for mast cell activation-toward the comprehensive prediction of cryptides acting on mast cells.

Mastoparan, a toxic peptide from wasp venom, induces various biological functions including histamine release from rat peritoneal mast cells. Here we report that, for the activation of mast cells by mastoparan, at least two positively charged side chains are required on the hydrophilic side of the amphiphilic structure of the peptide. The present results are expected to be utilized for the bioinformatic and comprehensive identification of endogenous mast cell-stimulating cryptides.

Discovery of posttranslational maturation by self-subunit swapping.

Several general mechanisms of metallocenter biosynthesis have been reported and reviewed, and in all cases, the components or subunits of an apoprotein remain in the final holoprotein. Here, we first discovered that one subunit of an apoenzyme did not remain in the functional holoenzyme. The cobalt-containing low-molecular-mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 consists of beta- and alpha-subunits encoded by the nhlBA genes, respectively.

Discovery of mitocryptide-1, a neutrophil-activating cryptide from healthy porcine heart.

Although neutrophils are known to migrate in response to various chemokines and complement factors, the substances involved in the early stages of their transmigration and activation have been poorly characterized to date. Here we report the discovery of a peptide isolated from healthy porcine hearts that activated neutrophils. Its primary structure is H-Leu-Ser-Phe-Leu-Ile-Pro-Ala-Gly-Trp-Val-Leu-Ser-His-Leu-Asp-His-Tyr-Lys-Arg-Ser-Ser-Ala-Ala-OH, and it was indicated to originate from mitochondrial cytochrome c oxidase subunit VIII.

Cryptide signaling: Amphiphilic peptide-induced exocytotic mechanisms in mast cells.

Amphiphilic peptides with positive charges such as substance P (SP) and mastoparan (MP) are known to induce exocytosis in rat peritoneal mast cells. To elucidate whether and how intracellular Ca(2+) signaling is involved in the peptide-induced exocytosis, here we investigated the relationships between an increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and exocytosis caused by SP and MP. SP and MP induced exocytosis coinciding with an initial rapid and transient [Ca(2+)](i) increase, but not with a sustained increase.

Transcriptional regulation of the nitrile hydratase gene cluster in Pseudomonas chlororaphis B23.

An enormous amount of nitrile hydratase (NHase) is inducibly produced by Pseudomonas chlororaphis B23 after addition of methacrylamide as the sole nitrogen source to a medium. The expression pattern of the P. chlororaphis B23 NHase gene cluster in response to addition of methacrylamide to the medium was investigated.

Discovery of amide (peptide) bond synthetic activity in Acyl-CoA synthetase.

Acyl-CoA synthetase, which is one of the acid-thiol ligases (EC 6.2.1), plays key roles in metabolic and regulatory processes.