Dipeptide Synthesis by an Aminopeptidase from Streptomyces septatus TH-2 and Its Application to Synthesis of Biologically Active Peptides

Dipeptide synthesis by aminopeptidase from Streptomyces septatus TH-2 (SSAP) was demonstrated using free amino acid as an acyl donor and aminoacyl methyl ester as an acyl acceptor in 98% methanol (MeOH). SSAP retained its activity after more than 100 h in 98% MeOH, and in the case of phenylalanyl-phenylalanine methyl ester synthesis, the enzyme reaction reached equilibrium when more than 50% of the free phenylalanine was converted to the product. In an investigation of the specificity of SSAP toward acyl donors and acyl acceptors, SSAP showed a broad specificity toward various free amino acids and aminoacyl methyl esters. Furthermore, we applied SSAP to the synthesis of several biologically active peptides, such as aspartyl-phenylalanine, alanyl-tyrosine, and valyl-tyrosine methyl esters.     

Change in Substrate Preference of Streptomyces Aminopeptidase through Modification of the Environment around the Substrate Binding Site

We attempted to alter the substrate preference of aminopeptidase from Streptomyces septatus TH-2 (SSAP). Because Asp198 and Phe221 of SSAP are located in the substrate binding site, we screened 2,000 mutant enzymes with D198X/F221X mutations. By carrying out this examination, we obtained two enzymes; one specifically hydrolyzed an arginyl derivative, and the other specifically hydrolyzed a cystinyl derivative (65- and 12.5-fold higher k_cat values for hydrolysis of p-nitroanilide derivatives than those of the wild type, respectively).     

Characterization of heme-coordinating histidyl residues of cytochrome b5 based on the reactivity with diethylpyrocarbonate: a mechanism for the opening of axial imidazole rings

We investigated the reactivity of heme-coordinating imidazole with diethylpyrocarbonate using a soluble domain of cytochrome b(5). Analyses with various spectroscopic methods including MALDI-TOF-MS indicated that two axial His residues (His44 and His68) of cytochrome b(5) were protected from the modification by several factors, i.e., limited steric exposure of the axial imidazole to the solvent, the Fe-N(epsilon2) coordination bond, and protonation of the N(delta1) position by forming a hydrogen bond with its immediate surroundings. However, once N-carbethoxylation at the N(epsilon2) position of the axial His residues occurred with a higher concentration of diethylpyrocarbonate, displacement of heme prosthetic group from the protein moiety continued. Simultaneously, it facilitated the second N-carbethoxylation to take place at the N(epsilon1) position of the same imidazole ring, leading to a bis-N-carbethoxylated derivative and further to a ring-opened derivative. A similar mechanism seemed in operation for one non-axial His residue (His85), in which the N(delta1) atom works as a hydrogen acceptor in a strong hydrogen-bond and the other N(epsilon2) atom is in a protonated form, resulting in a formation of the ring-opened derivative upon treatment with a higher concentration of diethylpyrocarbonate. These results suggested that the use of diethylpyrocarbonate for MALDI-TOF-MS analysis might provide a unique method to characterize the protonation state of His residues and the strength of their hydrogen-bondings at the active site of enzymes.     

The fine-scale genetic structure of the two-spotted spider mite in a commercial greenhouse

The fine-scale genetic structure of Tetranychus urticae Koch was studied to estimate local gene flow within a rose tree habitat in a commercial greenhouse using seven microsatellite markers. Two beds of rose trees with different population densities were selected and 18 consecutive quadrats of 1.2 m length were sequentially established in each bed. Heterozygote deficiency was positive within quadrats, which was most likely a result of the Wahlund effect because the mites usually form small breeding colonies. Low population density and frequent inbreeding could also accelerate genetic differentiation among the breeding colonies. A short-range (2.4–3.6 m) positive autocorrelation and clear genetic cline among quadrat populations was detected within a bed. This suggests that gene flow was limited to a short range even if population density was substantially increased. Therefore, large-scale dispersal such as aerial dispersal contributed very little to gene flow in the greenhouse.     

TNFalpha is required for cholestasis-induced liver fibrosis in the mouse

TNFα, a mediator of hepatotoxicity in several animal models, is elevated in acute and chronic liver diseases. Therefore, we investigated whether hepatic injury and fibrosis due to bile duct ligation (BDL) would be reduced in TNFα knockout mice (TNFα−/−). Survival after BDL was 60% in wild-type mice (TNFα+/+) and 90% in TNFα−/− mice. Body weight loss and liver to body weight ratios were reduced in TNFα−/− mice compared to TNFα+/+ mice. Following BDL, serum alanine transaminases (ALT) levels were elevated in TNFα+/+ mice (268.6 ± 28.2 U/L) compared to TNFα−/− mice (105.9 U/L ± 24.4). TNFα−/− mice revealed lower hepatic collagen expression and less liver fibrosis in the histology. Further, α-smooth muscle actin, an indicator for activated myofibroblasts, and TGF-β mRNA, a profibrogenic cytokine, were markedly reduced in TNFα−/− mice compared to TNFα+/+ mice. Thus, our data indicate that TNFα induces hepatotoxicity and promotes fibrogenesis in the BDL model.     

Biochemical characterization of a novel metalloendopeptidase from Streptomyces aureofaciens TH-3 with post-proline hydrolysis activity

Streptomyces aureofaciens TH-3 secretes a protease termed ‘kibilysin’, for which we showed unique substrate specificity and preference for Tyr, Pro, and Leu at the P₁ position using fluorescence energy transfer substrate (FRETS) combinatorial libraries. Using (7-methoxycoumarin-4-yl) acetyl-Lys-Pro-Leu-Gly-Leu-d-2,3-diamino propionic acid (2,4-dinitrophenyl)-Ala-Arg-NH₂, we confirmed that kibilysin digests the substrate between Pro and Leu. Its gene was cloned and sequenced. The primary structure of the enzyme showed 40, 66, and 61% identity, respectively, with those of thermolysin from Bacillus thermoproteolyticus, and metalloendopeptidases from Streptomyces cinamoneus TH-2 and S. griseus. Its deduced amino acid sequence contained an HEXXH consensus sequence for zinc binding, which is a common motif of the peptidase family M4. Moreover, we succeeded in over-expression of kibilysin using Streptomyces lividans.     

Influence of inbreeding depression on a lake population of Nymphoides peltata after restoration from the soil seed bank

The negative effects of inbreeding depression on fragmented small populations are likely to be expressed more strongly after restoration efforts if regeneration processes have been highly restricted in degraded habitats. We examined the potential influences of inbreeding depression on a population of Nymphoides peltata (Menyanthaceae) restored from the remnant soil seed bank. A hand-pollination experiment demonstrated self-compatibility of a single remaining homostyle genet and significant inbreeding depression in selfed progeny, especially in parameters related to seedling growth (–0.6 for biomass, and –0.4 for relative growth rate). Our genetic analysis indicated that the presumed number of parents contributing to the current soil seed bank was only 2–8 genets and that a single sib-family dominated at each of three sampling sites. The results also showed that the selfed progeny of the homostyle genet were overwhelmingly dominant at two sites (86.8 and 94.7%). As a result, the growth performance of the seed bank seedlings was significantly reduced, to a level as low as that of the selfed progeny. Active restoration efforts to minimize the negative effects of the genetic bottleneck and continuous monitoring based on genetic and demographic study are recommended.     

Structure, backbone dynamics and interactions with RNA of the C-terminal RNA-binding domain of a mouse neural RNA-binding protein, Musashi1

Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in the regulation of asymmetric cell division. Musashi1 contains two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2. Our previous studies showed that RBD1 alone binds to RNA, while the binding of RBD2 is not detected under the same conditions. Joining of RBD2 to RBD1, however, increases the affinity to greater than that of RBD1 alone, indicating that RBD2 contributes to RNA-binding. We have determined the three-dimensional solution structure of the C-terminal RBD (RBD2) of Musashi1 by NMR. It folds into a compact alpha beta structure comprising a four-stranded antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. Special structural features of RBD2 include a beta-bulge in beta2 and a shallow twist of the beta-sheet. The smaller 1H-15N nuclear Overhauser enhancement values for the residues of loop 3 between beta2 and beta3 suggest that this loop is flexible in the time-scale of nano- to picosecond order. The smaller 15N T2 values for the residues around the border between alpha2 and the following loop (loop 5) suggest this region undergoes conformational exchange in the milli- to microsecond time-scale. Chemical shift perturbation analysis indicated that RBD2 binds to an RNA oligomer obtained by in vitro selection under the conditions for NMR measurements, and thus the nature of the weak RNA-binding of RBD2 was successfully characterized by NMR, which is otherwise difficult to assess. Mainly the residues of the surface composed of the four-stranded beta-sheet, loops and C-terminal region are involved in the interaction. The appearance of side-chain NH proton resonances of arginine residues of loop 3 and imino proton resonances of RNA bases upon complex formation suggests the formation of intermolecular hydrogen bonds. The structural arrangement of the rings of the conserved aromatic residues of beta2 and beta3 is suitable for stacking interaction with RNA bases, known to be one of the major protein-RNA interactions, but a survey of the perturbation data suggested that the stacking interaction is not ideally achieved in the complex, which may be related to the weaker RNA-binding of RBD2.     

Identification of bioactive molecules by adipogenesis profiling of organic compounds

An important step in the postgenomic drug discovery is the construction of high quality chemical libraries that generate bioactive molecules at high rates. Here we report a cell-based approach to composing a focused library of biologically active compounds. A collection of bioactive non-cytotoxic chemicals was identified from a divergent library through the effects on the insulin-induced adipogenesis of 3T3-L1 cells, one of the most drastic and sensitive morphological alterations in cultured mammalian cells. The resulting focused library amply contained unique compounds with a broad range of pharmacological effects, including glucose-uptake enhancement, cytokine inhibition, osteogenesis stimulation, and selective suppression of cancer cells. Adipogenesis profiling of organic compounds generates a focused chemical library for multiple biological effects that are seemingly unrelated to adipogenesis, just as genetic screens with the morphology of fly eyes identify oncogenes and neurodegenerative genes.