Isao Kusakabe

Crystal structures of decorated xylooligosaccharides bound to a family 10 xylanase from Streptomyces olivaceoviridis E-86

The family 10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) consists of a GH10 catalytic domain, which is joined by a Gly/Pro-rich linker to a family 13 carbohydrate-binding module (CBM13) that interacts with xylan. To understand how GH10 xylanases and CBM13 recognize decorated xylans, the crystal structure of SoXyn10A was determined in complex with α-l-arabinofuranosyl- and 4-O-methyl-α-d-glucuronosyl-xylooligosaccharides. The bound sugars were observed in the subsites of the catalytic cleft and also in subdomains α and γ of CBM13. The data reveal that the binding mode of the oligosaccharides in the active site of the catalytic domain is entirely consistent with the substrate specificity and, in conjunction with the accompanying paper (Pell, G., Taylor, E. J., Gloster, T. M., Turkenburg, J. P., Fontes, C. M. G. A., Ferreira, L. M. A., Nagy, T., Clark, S. J., Davies, G. J., and Gilbert, H. J. (2004) J. Biol. Chem. 279, 9597-9605), demonstrate that the accommodation of the side chains in decorated xylans is conserved in GH10 xylanases of SoXyn10A against arabinoglucuronoxylan. CBM13 was shown to bind xylose or xylooligosaccharides reversibly by using nonsymmetric sugars as the ligands. The independent multiple sites in CBM13 may increase the probability of substrate binding.

Preparation of (1→4)-β-d-xylooligosaccharides from an acid hydrolysate of cotton-seed xylan: suitability of cotton-seed xylan as a starting material for the preparation of (1→4)-β-d-xylooligosaccharides

Cotton-seed residual cake, which is a byproduct of the process of oil extraction from the seed, was delignified with sodium hypochlorite (1% available chlorine). Xylan was then prepared from the delignified wet material by alkali extraction with 15% sodium hydroxide. The cotton-seed xylan contained 64.7% xylose and 9.4% uronic acid. The xylan was hydrolyzed with 0.125 M sulfuric acid at 90 degrees C for 15 min. The resultant hydrolysis products were separated by gel-permeation chromatography on BioGel P-4 and Toyopearl HW-40F columns connected in series, with water as an eluate. Xylose and xylooligosaccharides with a degree of polymerization ranging from DP 2 to 15 were separated under such conditions, and each xylooligosaccharide-containing peak fraction afforded a single band on fluorophore-assisted carbohydrate electrophoresis. These results suggest that cotton-seed xylan is suitable for the preparation of xylose and xylooligosaccharides.

Potent angiogenic inhibition effects of deacetylated chitohexaose separated from chitooligosaccharides and its mechanism of action in vitro

This study was performed to demonstrate the effects of deacetylated chitohexaose (hexamer) separated from a chitooligosaccharide (COS) mixture on tumor angiogenesis and its mechanism of action. Five fractions from dimer to hexamer were separated by a linear gradient solution of HCl on a cation-exchange resin. Then HCl was removed from the fractions by a charcoal column. The purity of the five fractions was analyzed by HPLC and the molecular masses were analyzed by MALDI-TOFMS. The hexamer expressed an inhibitory influence on CAM angiogenesis in a dose-dependent manner at concentrations of 6.25-50microg/egg. On further investigation, we found that the hexamer had no toxic effect on normal ECV304 cells, but could inhibit the proliferation and migration of tumor-induced ECV304 cells in a dose-dependent manner. The mechanism was demonstrated through the detection of mRNA expression of VEGF, MMP-9, TIMP-1, TIMP-2, and uPA by RT-PCR, which showed that the hexamer down-regulated the VEGF and uPA mRNA expressions in ECV304 cells, but up-regulated the TIMP-1 mRNA expression.

PCR cloning and expression of the F/10 family xylanase gene from Streptomyces olivaceoviridis E-86

Abstract Using a simple long-range inverse PCR method, we cloned the GC-rich gene (68%) for an F 10 xylanase from Streptomyces olivaceoviridis E-86. The open reading frame of the cloned gene, fxyn, contained 1431 bp and encoded 477 amino acid residues. FXYN resembled a xylanase of the F 10 family and had two functional domains (a catalytic domain and a substrate-binding domain). Unique triple repeat sequence regions (CLD-C) were located in the substrate-binding domain, which was similar to the xylan-binding domains of xylanase A and that of arabinofuranosidase B from S. lividans. FXYN with a tag that consisted of six histidine residues at the carboxy-terminus was expressed at high levels in Escherichia coli and had the same properties as the native xylanase produced by S. olivaceoviridis. Moreover, the xylan-binding domain of FXYN significantly enhanced hydrolysis of insoluble xylan whereas it had minimal effect on the hydrolysis of soluble xylan.

α-Galactosidase from cultured rice (Oryza sativa L. var. Nipponbare) cells

The alpha-galactosidase from rice cell suspension cultures was purified to homogeneity by different techniques including affinity chromatography using N-epsilon-aminocaproyl-alpha-D-galactopyranosylamine as the ligand. From 11 l of culture filtrate, 28.7 mg of purified enzyme was obtained with an overall yield of 51.9%. The cDNA coding for the alpha-galactosidase was cloned and sequenced. The enzyme was found to contain 417 amino acid residues composed of a 55 amino acid signal sequence and 362 amino acid mature alpha-galactosidase; the molecular weight of the mature enzyme was thus calculated to be 39,950. Seven cysteine residues were also found but no putative N-glycosylation sites were present. The observed homology between the deduced amino acid sequences of the mature enzyme and alpha-galactosidases from coffee (Coffea arabica), guar (Cyamopsis tetragonolooba), and Mortierella vinacea alpha-galactosidase II were over 73, 72, and 45%, respectively. The enzyme displayed maximum activity at 45 degrees C when p-nitrophenyl-alpha-D-galactopyranoside was used as substrate. The rice alpha-galactosidase and Mortierella vinacea alpha-galactosidase II acted on both the terminal alpha-galactosyl residue and the side-chain alpha-galactosyl residue of the galactomanno-oligosaccharides.

Purification and Characterization of the Recombinant Thermus sp. Strain T2 α-Galactosidase Expressed in Escherichia coli

ABSTRACT The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable α-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (Mr, 53,514). The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus brockianus was over 70%.Thermus sp. strain T2 α-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75°C forp-nitrophenyl-α-d-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70°C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40°C for 1 h. The enzyme acted on the terminal α-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea α-galactosidase I. The enzyme has only one Cys residue in the molecule.para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.

Structure of rice-straw arabinoglucuronoxylan and specificity of Streptomyces xylanase toward the xylan.

Rice-straw treated with 5% ammonia water was hydrolyzed with the beta-xylanase of Streptomyces sp. E-86, and four kinds of hetero-oligosaccharides, two arabinoxylo- and two glucuronoxylo-oligosaccharides, were isolated from the hydrolysate by charcoal column, gel filtration, ion-exchange, and aminopropyl silica column chromatographies. The structures of the oligosaccharides were identified as 3(2)-alpha-L-arabinofuranosyl-xylobiose, 3(2)-alpha-L-arabinofuranosylxylotriose, 2(3)-4-O-methyl-alpha-D-glucuronosylxylotriose, and 2(3)-alpha-D-glucuronosylxylotriose by the analysis of component sugar, partial acid hydrolysis, methylation analysis etc. Based on the structures of above oligosaccharides, the structure of rice-straw arabinoglucuronoxylan and the specificity of the xylanase toward the xylan are discussed.

Synthesis of regioisomeric methyl α-L-arabinofuranobiosides

Abstract The three regioisomers of methyl α- l - arabinofuranobioside , namely methyl O-α- l - arabinofuranosyl -(1 → 3)-α- l - arabinofuranoside , methyl O-α- l - arabinofuranosyl -(1 → 3)-α- l - arabinofuranoside , and methyl O-α- l - arabinofuranosyl -(1 → 5)-α- l - arabinofuranoside , were synthesized for use as substrates in studies of the specificity of α- l - arabinofuranosidase . The regiospecifically protected precursors, namely methyl 3,5- di -O- benzoyl -α- l - arabinofuranoside,methyl 2,5-di -O- benzyl -α- l - arabinofuranoside , and methyl 2,3- di -O- benzoyl -α- l - arabinofuranoside , were prepared from 2,3,5- tri -O- benzoyl -α- l - arabinofuranosyl chloride ( 4 ) and methyl 5-O- trityl -α- l - arabinofuranoside , respectively, and glycosylated with 4 in the presence of silver trifluoromethanesulfonate and s -collidine. 1 H and 13 C NMR data for all compounds are presented.

Purification and characterization of aspartic proteinase from sunflower seeds

Aspartic proteinases were purified from sunflower seed extracts by affinity chromatography on a pepstatin A-EAH Sepharose column and by Mono Q column chromatography. The final preparation contained three purified fractions. SDS-PAGE showed that one of the fractions consisted of disulfide-bonded subunits (29 and 9 kDa), and the other two fractions contained non-covalently bound subunits (29 and 9 kDa). These purified enzymes showed optimum pH for hemoglobinolytic activity at pH 3.0 and were completely inhibited by pepstatin A like other typical aspartic proteinases. Sunflower enzymes showed more restricted specificity on oxidized insulin B chain and glucagon than other aspartic proteinases. The cDNA coding for an aspartic proteinase was cloned and sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 440 amino acid residues with a molecular mass of 47,559 Da. The difference between the molecular size of purified enzymes and of the mature enzyme was due to the fact that the purified enzymes were heterodimers formed by the proteolytic processing of the mature enzyme. The derived amino acid sequence of the enzyme showed 30-78% sequence identity with that of other aspartic proteinases.

An investigation of the nature and function of module 10 in a family F/10 xylanase FXYN of Streptomyces olivaceoviridis E-86 by module shuffling with the Cex of Cellulomonas fimi and by site-directed mutagenesis

Although the amino acid homology in the catalytic domain of FXYN xylanase from Streptomyces olivaceoviridis E‐86 and Cex xylanase from Cellulomonas fimi is only 50%, an active chimeric enzyme was obtained by replacing module 10 in FXYN with module 10 from Cex. In the family F/10 xylanases, module 10 is an important region as it includes an acid/base catalyst and a substrate binding residue. In FXYN, module 10 consists of 15 amino acid residues, while in Cex it consists of 14 amino acid residues. The K m and k cat values of the chimeric xylanase FCF‐C10 for PNP‐xylobioside (PNP‐X2) were 10‐fold less than those for FXYN. CD spectral data indicated that the structure of the chimeric enzyme was similar to that of FXYN. Based on the comparison of the amino acid sequences of FXYN and Cex in module 10, we constructed four mutants of FXYN. When D133 or S135 of FXYN was deleted, the kinetic properties were not changed from those of FXYN. By deletion of both D133 and S135, the K m value for PNP‐X2 decreased from the 2.0 mM of FXYN to 0.6 mM and the k cat value decreased from the 20 s−1 of FXYN to 8.7 s−1. Insertion of Q140 into the doubly deleted mutant further reduced the K m value to 0.3 mM and the k cat value to 3.8 s−1. These values are close to those for the chimeric enzyme FCF‐C10. These results indicate that module 10 itself is able to accommodate changes in the sequence position of amino acids which are critical for enzyme function. Since changes of the spatial position of these amino acids would be expected to result in enzyme inactivation, module 10 must have some flexibility in its tertiary structure. The structure of module 10 itself also affects the substrate specificity of the enzyme.