Shinya Sasaki

Lignin dehydrogenative polymerization mechanism : a poplar cell wall peroxidase directly oxidizes polymer lignin and produces in vitro dehydrogenative polymer rich in β-O-4 linkage

An investigation was performed to determine whether lignin dehydrogenative polymerization proceeds via radical mediation or direct oxidation by peroxidases. It was found that coniferyl alcohol radical transferred quickly to sinapyl alcohol. The transfer to syringaresinol was slower, however, the transfer to polymeric lignols occurred very slightly. This result suggests that the radical mediator theory does not sufficiently explain the mechanism for dehydrogenative polymerization of lignin. A cationic cell wall peroxidase (CWPO‐C) from poplar (Populus alba L.) callus showed a strong substrate preference for sinapyl alcohol and the sinapyl alcohol dimer, syringaresinol. Moreover, CWPO‐C was capable of oxidizing high‐molecular‐weight sinapyl alcohol polymers and ferrocytochrome c. Therefore, the CWPO‐C characteristics are important to produce polymer lignin. The results suggest that CWPO‐C may be a peroxidase isoenzyme responsible for the lignification of plant cell walls.

Sinapyl alcohol-specific peroxidase isoenzyme catalyzes the formation of the dehydrogenative polymer from sinapyl alcohol

Two peroxidases, CWPO-A and CWPO-C, were isolated from the cell walls of poplar (Populus alba L.) callus culture. The cationic CWPO-C showed a strong preference for sinapyl alcohol over coniferyl alcohol as substrate. Thus, the monolignol utilization of CWPO-C is unique compared with other peroxidases, including anionic CWPO-A and horseradish peroxidase (HRP). CWPO-C polymerized oligomeric sinapyl alcohol (S-oligo) and sinapyl alcohol, producing a polymer of greater molecular weight. In contrast, HRP, which is specific to coniferyl alcohol, produced sinapyl alcohol dimers, rather than catalyzing polymerization. Adding coniferyl alcohol as a radical mediator in the HRP-mediated reaction did not result in S-oligo polymerization. This report shows that CWPO-C is an isoenzyme specific to sinapyl alcohol that polymerizes oligomeric lignols. Its catalytic activity toward oligomeric lignols may be related to the lignification of angiosperm woody plant cell walls.

The cationic cell-wall-peroxidase having oxidation ability for polymeric substrate participates in the late stage of lignification of Populus alba L

Previously we reported that purified Cell Wall Peroxidase-Cationic (CWPO-C) from poplar callus (Populus alba L.) oxidizes sinapyl alcohol and polymeric substrate unlike other plant peroxidases and proposed that this isoenzyme is a conceivable lignification specific peroxidase. In this study, we cloned full-length cDNA of CWPO-C and investigated the transcription of CWPO-C gene in various organs and the localization of CWPO-C protein in the differentiating xylem of poplar stem.Real-time PCR analyses indicated that CWPO-C gene is constitutively expressed in the developing xylem, leaf, and shoot but not affected by many stress treatments. Immunohistochemical analysis showed that CWPO-C locates in the middle lamellae, cell corners, and secondary cell walls of the fiber cells during the lignification. The intensity of the CWPO-C labeling increased gradually from the cell wall thickening stage to mature stage of fiber cells, which is very consistent with the increase of lignin content in the developing xylem. These results strongly support that CWPO-C is responsible for the lignification of the secondary xylem. Interestingly, immuno-labeling of CWPO-C was also observed inside of the ray parenchyma cells instead no signals were detected within the developing fiber cells. This suggests that CWPO-C is biosynthesized in the parenchyma cells and provided to the middle lamellae, the cell corners, and the cell walls to achieve lignin polymerization.

Transcriptional and translational analyses of poplar anionic peroxidase isoenzymes

Anionic peroxidases have been proposed to be a key enzyme for lignification in poplar. On the other hand, there are many genes encoding an anionic peroxidase in Populus trichocarpa genome, and their physiological functions are still unknown. Ampholine isoelectric focusing analysis revealed anionic peroxidases as dominant peroxidases in enzyme preparations from various organs. Using two-dimensional electrophoresis (2-DE) followed by peptide mass fingerprint (PMF) analysis, we surveyed the localization of anionic peroxidase isoenzymes in various organs of Populus alba L. Peroxidase isoenzymes were extracted from various organs and fractionated by a Concanavallin A Sepharose column. Each protein was separated by 2-DE gels and some anionic peroxidase isoenzymes in each organ were identified via PMF analysis. Transcript and protein of individual peroxidase indicate that the expression profile of each isoenzyme is quite different, for example, organspecific gene, stress-response gene, and multifunction gene, even though they are in the same cluster. These results suggest that individual anionic isoenzymes in this small cluster were differently regulated at transcription, translation, or posttranslation.

Hydrophobicity of water at the surface as studied by laser-induced fluorescence microscopy

A sensitive comparative fluorescence microscopic approach was developed and used to study the water at the air-water interface region. An insoluble chromophore mesoc-α,β,γ,δ-tetraphenylporphine (TPP) was spread on the water. Its fluorescence spectrum indicated that the spectrum of TPP on the water surface was similar to those measured in nonpolar hydrocarbon solvents. Thus, the water at the surface was concluded to be hydrophobic and less polar than the water in the bulk.

Enzymatic activities for lignin monomer intermediates highlight the biosynthetic pathway of syringyl monomers in Robinia pseudoacacia.

Most of the known 4-coumarate:coenzyme A ligase (4CL) isoforms lack CoA-ligation activity for sinapic acid. Therefore, there is some doubt as to whether sinapic acid contributes to sinapyl alcohol biosynthesis. In this study, we characterized the enzyme activity of a protein mixture extracted from the developing xylem of Robinia pseudoacacia. The crude protein mixture contained at least two 4CLs with sinapic acid 4-CoA ligation activity. The crude enzyme preparation displayed negligible sinapaldehyde dehydrogenase activity, but showed ferulic acid 5-hydroxylation activity and 5-hydroxyferulic acid O-methyltransferase activity; these activities were retained in the presence of competitive substrates (coniferaldehyde and 5-hydroxyconiferaldehyde, respectively). 5-Hydroxyferulic acid and sinapic acid accumulated in the developing xylem of R. pseudoacacia, suggesting, in part at least, sinapic acid is a sinapyl alcohol precursor in this species.

Synthesis and Photochemical Behaviour of 3-(Estran-16-yl)acrylates and 2-(Estran-16-yl)vinyl Ketones

C-16-substituted steroids having an unsaturation in the side chain have been synthesized by sequential Arnold–Vilsmeier and Wittig reactions, subsequent photochemical studies showing the formation of either a dimeric structure or the occurrence of E/Z-isomerization; for one example, treatment with H 2 over Pd–C led to full reduction of the side-chain and ring D.