Production of 2-phenylethanol in roses as the dominant floral scent compound from L-phenylalanine by two key enzymes, a PLP-dependent decarboxylase and a phenylacetaldehyde reductase

We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[(2)H8] Phenylalanine was converted to [(2)H8] phenylacetaldehyde and [(2)H8]-2-phenylethanol by two enzymes derived from the flower petals of R. 'Hoh-Jun,' these being identified as pyridoxal-5'-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH3 together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.     

Comparative proteome analysis of changes in the 26S proteasome during oocyte maturation in goldfish

Proteasomes are large, multi-subunit particles that act as the proteolytic machinery for most of the regulated intracellular protein degradation in eukaryotic cells. An alteration of proteasome function may be important for the regulation of the meiotic cell cycle. To study the change at the subunit level of the 26S proteasome during meiotic maturation, we purified 26S proteasomes from immature and mature oocytes of goldfish. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. For differential analysis, whole spots of the 26S proteasome from goldfish oocytes were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database analysis. Four spots that were different (only detected in mature oocyte 265 proteasomes and not in immature ones) and four protein spots that were up- or down-regulated were identified unambiguously. The mature-specific spots were not 26S proteasome components but rather their interacting proteins, and were identified as chaperonin-containing TCP-1 subunits and myosin light chain. Minor spots of three subunits of the 20S core particle and one of the 19S regulatory particle showed meiotic cell cycle-dependent changes. These results demonstrate that modifications of proteasomal subunits and cell cycle phase-dependent interactions of proteins with proteasomes occur during oocyte maturation in goldfish.     

Mannose-specific lectin from the mushroom Hygrophorus russula

A lectin was purified from the mushroom Hygrophorus russula by affinity chromatography on a Sephadex G-50 column and BioAssist S cation exchange chromatography and designated H. russula lectin (HRL). The results of sodium dodecyl sulfate-polyaclylamidegel electrophoresis, gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of HRL indicated that it was composed of four identical 18.5?kDa subunits with no S-S linkage. Isoelectric focusing of the lectin showed bands near pI 6.40. The complete sequence of 175 amino acid residues was determined by amino acid sequencing of intact or enzyme-digested HRL. The sequence showed homology with Grifola frondosa lectin. The cDNA of HRL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA consisted of 528?bp encoding 176 amino acids. In hemagglutination inhibition assay, α1-6 mannobiose was the strongest inhibitor and isomaltose, Glcα1-6Glc, was the second strongest one, among mono- and oligosaccharides tested. Frontal affinity chromatography indicated that HRL had the highest affinity for Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc, and non-reducing terminal Manα1-6 was essential for the binding of HRL to carbohydrate chains. The sugar-binding specificity of HRL was also analyzed by using BIAcore. The result from the analysis exhibited positive correlations with that of the hemagglutination inhibition assay. All the results suggested that HRL recognized the α1-6 linkage of mannose and glucose, especially the Manα1-6 bond. HRL showed a mitogenic activity against spleen lymph cells of an F344 rat. Furthermore, an enzyme-linked immunosorbent assay showed strong binding of HRL to human immunodeficiency virus type-1 gp120.     

The effects of gene disruption of Kre6-like proteins on the phenotype of β-glucan-producing Aureobasidium pullulans

Applied Microbiology and Biotechnology - Killer toxin resistant 6 (Kre6) and its paralog, suppressor of Kre null 1 (Skn1), are thought to be involved in the biosynthesis of cell wall...     

Purification and characterization of beta-glucosidase involved in the emission of 2-phenylethanol from rose flowers

Beta-glucosidase was partially purified from Rosa 'Hoh-Jun' petals. The enzyme was highly specific for such beta-D-glucopyranosides as 2-phenylethyl beta-D-glucopyranoside. The optimal activity was observed at pH 6.0 and 35 degrees C. The enzymes were composed with two proteins (160 and 155 kDa) by blue native-PAGE, and were classified in a family 1 glucosidase based on LC-MS/MS analyses.     

Purification, characterization, and sugar binding specificity of an N-Glycolylneuraminic acid-specific lectin from the mushroom Chlorophyllum molybdites

A carbohydrate-binding protein was isolated from the carpophores of the mushrooms and designated the Chlorophyllum molybdites lectin (CML) based on its origin. The molecular mass of CML was 32 kDa, and it was composed of two 16-kDa monomers with no disulfide bonds. Monosaccharide analysis indicated that 12% of the mass of CML was carbohydrate and consisted of GlcNAc:GalNAc:Gal:Man:l-Fuc in a molar ratio of 1.5:1.9: 4.4:4.8:1.0. In the hemagglutination inhibition assay, CML exhibited the strongest binding specificity toward N-glycolylneuraminic acid (NeuGc) among the monosaccharides tested, whereas NeuAc did not inhibit the hemagglutination at all. GalNAc and Mealpha-GalNAc were also inhibitory at much higher concentrations than NeuGc. Among the glycoproteins, asialobovine submaxillary mucin (BSM) and porcine stomach mucin (PSM) showed strong inhibitory effects. In surface plasmon resonance analysis, asialo-BSM and PSM exhibited the strongest binding affinity. After co-injection of CML and NeuGc or GalNAc onto the asialo-BSM- or PSM-immobilized chip, the dissociation of CML from the immobilized PSM was accelerated by NeuGc and GalNAc, but the dissociation of CML from the immobilized asialo-BSM was only promoted by GalNAc. These results and the other surface plasmon resonance experiments allowed us to conclude that the binding of asialo-BSM to CML was because of an interaction between the lectin and the GalNAc residues of asialo-BSM, and both the NeuGc and GalNAc residues were responsible for the binding of PSM to CML. The results also suggested that CML had two different carbohydrate binding domains, one specific for NeuGc and the other for GalNAc.     

A Novel Core Fucose-specific Lectin from the Mushroom Pholiota squarrosa

Fucα1–6 oligosaccharide has a variety of biological functions and serves as a biomarker for hepatocellular carcinoma because of the elevated presence of fucosylated α-fetoprotein (AFP) in this type of cancer. In this study we purified a novel Fucα1–6-specific lectin from the mushroom Pholiota squarrosa by ion-exchange chromatography and affinity chromatography on thyroglobulin-agarose. The purified lectin was designated as PhoSL (P. squarrosa lectin). SDS-PAGE, MALDI-TOF mass spectrometry, and N-terminal amino acid sequencing indicate that PhoSL has a molecular mass of 4.5 kDa and consists of 40 amino acids (NH₂-APVPVTKLVCDGDTYKCTAYLDFGDGRWVAQWDTNVFHTG-OH). Isoelectric focusing of the lectin showed bands near pI 4.0. The lectin activity was stable between pH 2.0 and 11.0 and at temperatures ranging from 0 to 100 °C for incubation times of 30 min. When PhoSL was investigated with frontal affinity chromatography using 132 pyridylaminated oligosaccharides, it was found that the lectin binds only to core α1–6-fucosylated N-glycans and not to other types of fucosylated oligosaccharides, such as α1–2-, α1–3-, and α1–4-fucosylated glycans. Furthermore, PhoSL bound to α1–6-fucosylated AFP but not to non-fucosylated AFP. In addition, PhoSL was able to demonstrate the differential expression of α1–6 fucosylation between primary and metastatic colon cancer tissues. Thus, PhoSL will be a promising tool for analyzing the biological functions of α1–6 fucosylation and evaluating Fucα1–6 oligosaccharides as cancer biomarkers.