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.     

Multiple points of interaction between retinoic acid and FGF signaling during embryonic axis formation

Anteroposterior (AP) patterning of the developing CNS is crucial for both regional specification and the timing of neurogenesis. Several important factors are involved in AP patterning, including members of the WNT and FGF growth factor families, retinoic acid receptors, and HOX genes. We have examined the interactions between FGF and retinoic signaling pathways. Blockade of FGF signaling downregulates the expression of members of the RAR signaling pathway, RARalpha, RALDH2 and CYP26. Overexpression of a constitutively active RARalpha2 rescues the effects of FGF blockade on the expression of XCAD3 and HOXB9. This suggests that RARalpha2 is required as a downstream target of FGF signaling for the posterior expression of XCAD3 and HOXB9. Surprisingly, we found that posterior expression of FGFR1 and FGFR4 was dependent on the expression of RARalpha2. Anterior expression was also altered with FGFR1 expression being lost, whereas FGFR4 expression was expanded beyond its normal expression domain. RARalpha2 is required for the expression of XCAD3 and HOXB9, and for the ability of XCAD3 to induce HOXB9 expression. We conclude that RARalpha2 is required at multiple points in the posteriorization pathway, suggesting that correct AP neural patterning depends on a series of mutually interactive feedback loops among FGFs, RARs and HOX genes.     

Asperyellone,a New Yellow Pigment of Aspergillus awamori and Aspergillus niger

Structure (VII), 7-methyl-13-phenyl-3-oxo-trideca-4, 6, 8, 10, 12-pentaene, was suggested for asperyellone, a yellow pigment isolated from mycelium of Aspergillus awamori 22–2–2.     

Identification and quantification of brassinolide-related steroids in the insect gall and healthy tissues of the chestnut plant

Castasterone and 6-deoxocastasterone were identified by GC/MS and/or selected ion monitoring not only in the insect gall of chestnut but also in the healthy tissues, including the shoot, leaf and flower bud. In addition, the gall was found to contain a small amount of brassinolide. Brassinone, which had been reported to be present in the gall, could not be detected in these tissues. The contents of castasterone, 6-deoxocastasterone and brassinolide, which were determined by using selected ion monitoring, are discussed.     

Bacteriophage-induced lytic enzyme which hydrolyzes L-alanine-D-glutamic acid peptide bond in peptidoglycan

The mode of action of bacteriophage-induced lytic enzyme “LE95” was investigated. The LE95 hydrolyzed peptide portion in peptidoglycan of $\text{Ps. aeruginosa}$ and $\text{E. coli}$. The exposed amino terminal amino acid was identified as glutamic acid by analysis of terminal amino acid by dinitrophenylation. This result suggested the LE95 hydrolyzed the peptide bond between L-alanine and D-glutamic acid in the peptidoglycan of $\text{Ps. aeruginosa}$ and $\text{E. coli}$. The enzyme did not hydrolyze various peptides prepared from bacterial cell wall. This experimental result suggested that the glycan chain of peptidoglycan would be essential for the enzymic activity.     

Crystal Structure of Cucumisin,a Subtilisin-Like Endoprotease from Cucumis melo L.

Cucumisin is a plant serine protease, isolated as an extracellular glycoprotein from the melon fruit Cucumis melo L. var. Prince. Cucumisin is composed of multiple domain modules, including catalytic, protease-associated, and fibronectin‐III-like domains. The crystal structure of cucumisin was determined by the multiwavelength anomalous dispersion method and refined at 2.75 Å resolution. A structural homology search indicated that the catalytic domain of cucumisin shares structural similarity with subtilisin and subtilisin-like fold enzymes. According to the Z-score, the highest structural similarity is with tomato subtilase 3 (SBT3), with an rmsd of 3.5 Å for the entire region. The dimer formation mediated by the protease-associated domain in SBT3 is a distinctive structural characteristic of cucumisin. On the other hand, analytical ultracentrifugation indicated that cucumisin is mainly monomeric in solution. Although the locations of the amino acid residues composing the catalytic triad are well conserved between cucumisin and SBT3, a disulfide bond is uniquely located near the active site of cucumisin. The steric circumstances of the active site with this disulfide bond are distinct from those of SBT3, and it contributes to the substrate preference of cucumisin, especially at the P2 position. Among the plant serine proteases, the thermostability of cucumisin is higher than that of its structural homologue SBT3, as determined by their melting points. A structural comparison between cucumisin and SBT3 revealed that cucumisin possesses less surface area and shortened loop regions. Consequently, the higher thermostability of cucumisin is achieved by its more compact structure.     

Genetic mapping of natural variation in potassium concentrations in shoots of Arabidopsis thaliana

Naturally-occurring variation in K(+) concentrations between plant genotypes is potentially exploitable in a number of ways, including altering the relationship between K(+) accumulation and growth, enhancing salinity resistance, or improving forage quality. However, achieving these requires greater insight into the genetic basis of the variation in tissue K(+) concentrations. To this end, K(+) concentrations were measured in the shoots of 70 Arabidopsis thaliana accessions and a Cape Verdi Island/Landsberg erecta recombinant inbred line (RIL) population. The shoot K(+) concentrations expressed on the basis of fresh matter (KFM) or dry matter (KDM) were both broadly and normally distributed as was the shoot dry matter content per unit fresh weight (DMC). Using the data from the RILs, four quantitative trait loci (QTL) were identified for KFM and three for KDM. These were located on chromosomes 2, 3, 4, and 5. Two of the QTLs for KFM overlapped with those for KDM. None of these QTLs overlapped with those for fresh weight or dry weight, but the QTL for KDM located on chromosome 3 overlapped with one for DMC. In silico analysis was used to identify known or putative K(+) and cation transporter genes whose loci overlapped with the QTLs. In most cases, multiple genes were identified and the possible role of their gene products in determining shoot K(+) concentrations is discussed.     

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).