Biomedical subjects

Junji Sugiyama

Publications and source records attributed to Junji Sugiyama.

6 recordsLinked to original sources

PubMed2003

Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin.

Bacillus circulans chitinase A1 (ChiA1) has a deep substrate-binding cleft on top of its (beta/alpha)8-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested. To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out. Y56A and W53A mutations at subsites -5 and -3, respectively, selectively decreased the hydrolysing activity against highly crystalline beta-chitin. W164A and W285A mutations at subsites +1 and +2, respectively, decreased the hydrolysing activity against crystalline beta-chitin and colloidal chitin, but enhanced the activities against soluble substrates. These mutations increased the K(m)-value when reduced (GlcNAc)5 (where GlcNAc is N -acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiA1 at higher concentrations of this substrate. In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite -1 decreased the hydrolysing activity drastically against all substrates and reduced the kcat-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively. From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis. W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates. W433 and Y279 are both essential for catalytic reaction as predicted from the structure.

Amino Acids, Aromatic↗

PubMed2003

The directionality of chitin biosynthesis: a revisit.

The molecular directionality of chitin biosynthesis was investigated by transmission electron microscopy (TEM) using electron crystallography methods applied to reducing-end-labelled beta-chitin microcrystals from vestimentiferan Lamellibrachia satsuma tubes and nascent beta-chitin microfibrils from the diatom Thalassiosira weissflogii. The data allowed confirmation that the microfibrils were extruded with their reducing end away from the biosynthetic loci, an orientation consistent only with elongation through polymerization at the non-reducing end of the growing chains. Such a chain-extension mechanism, which has also been demonstrated for cellulose and hyaluronan, appears to be general for glycosyltransferases that belong to the GT2 (glycosyl transferase 2) family. The data also allowed confirmation that in beta-chitin the chains are crystallized in a 'parallel-up' mode, in contrast with hypotheses proposed in previous reports.

Animals↗

PubMed2003

The binding specificity and affinity determinants of family 1 and family 3 cellulose binding modules.

Cellulose binding modules (CBMs) potentiate the action of cellulolytic enzymes on insoluble substrates. Numerous studies have established that three aromatic residues on a CBM surface are needed for binding onto cellulose crystals and that tryptophans contribute to higher binding affinity than tyrosines. However, studies addressing the nature of CBM-cellulose interactions have so far failed to establish the binding site on cellulose crystals targeted by CBMs. In this study, the binding sites of CBMs on Valonia cellulose crystals have been visualized by transmission electron microscopy. Fusion of the CBMs with a modified staphylococcal protein A (ZZ-domain) allowed direct immuno-gold labeling at close proximity of the actual CBM binding site. The transmission electron microscopy images provide unequivocal evidence that the fungal family 1 CBMs as well as the family 3 CBM from Clostridium thermocellum CipA have defined binding sites on two opposite corners of Valonia cellulose crystals. In most samples these corners are worn to display significant area of the hydrophobic (110) plane, which thus constitutes the binding site for these CBMs.

Base Sequence↗

PubMed2003

Enhancement of growth by expression of poplar cellulase in Arabidopsis thaliana.

To study the role of cellulose and cellulase in plant growth, we expressed poplar cellulase (PaPopCel1) constitutively in Arabidopsis thaliana. Expression increased the size of the rosettes due to increased cell size. The change in growth was accompanied by changes in biomechanical properties due to cell wall structure indicative of decrease in xyloglucan cross-linked with cellulose microfibrils by chemical analysis and nuclear magnetic resonance (NMR) spectra. The result supports the concept that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase to loosen xyloglucan intercalation and this irreversible wall modification promotes the enlargement of plant cells.

Arabidopsis↗

PubMed2002

Directional degradation of beta-chitin by chitinase A1 revealed by a novel reducing end labelling technique.

A novel procedure for labelling the molecular ends of beta-chitin crystals has been established. By introducing a hydrazide derivative of biotin at the reducing end of a chitin chain, followed by a specific interaction between biotin and streptavidin coupled with a colloidal gold particle, the chain directionality of beta-chitin microcrystals could be directly visualized by transmission electron microscopy. This method allowed to certify the parallelism of the chitin chains in the beta-chitin microcrystals, and also to label the reducing tips of beta-chitin microcrystals degraded by Bacillus circulans chitinase A1. With these substrates, the labelling occurred only at their tapered tip, which indicates that the digestion of these crystals proceeded from their reducing end. The generalization of this new labelling method to other polysaccharide crystals is discussed.

Animals↗

PubMedDate not supplied

X-ray microbeam and electron diffraction experiments on developing xylem cell walls.

This paper describes the first successful application of the novel technique of X-ray microbeam diffraction to the study of wood cell walls in developing xylem tissue. The method enabled us to obtain quantitative diffraction diagrams from single cell walls. It was further combined with selected area electron diffraction. We find that the crystal size of cellulose is increasing from the primary wall (P, <19 A) over the outer layer (S(1), 19 A) to the middle layer of the secondary wall (S(2), 24 A). In particular, the cellulose crystals formed in the primary wall consist of an extremely low-crystalline state of cellulose I. We suggest the more applicable concept of microfibrils with increasing lateral disorder, similar to cellulose IV(I).

Cell Wall↗