Identification of the gene structure and promoter region of H+-translocating inorganic pyrophosphatase in rice (Oryza sativa L. ).

In order to determine the gene structure and promoter region of vacuolar H+-translocating inorganic pyrophosphatase (V-PPase), we isolated the genomic clones using a rice BAC library and probes derived from rice V-PPase cDNA (OVP1). The entire OVP1 gene is approx. 5.4 kb in length, and seven introns interrupt the coding sequence of OVP1. The first intron is extremely large (1869 bp), while the other introns are between 82 and 170 bp. A transcription initiation site, identified by a primer extension analysis, indicated the first exon to be 366 bp. A 1.1 kb fragment containing the 5'-flanking region of the first exon with the GUS reporter gene showed specific promoter activity in rice cells. These data show that the OVP1 gene is composed of eight exons and seven introns, and regulatory elements are present within 1.1 kb upstream from the first exon.

Crystal structure of Streptomyces olivaceoviridis E-86 beta-xylanase containing xylan-binding domain.

Xylanases hydrolyse the beta-1,4-glycosidic bonds within the xylan backbone and belong to either family 10 or 11 of the glycoside hydrolases, on the basis of the amino acid sequence similarities of their catalytic domains. Generally, xylanases have a core catalytic domain, an N and/or C-terminal substrate-binding domain and a linker region. Until now, X-ray structural analyses of family 10 xylanases have been reported only for their catalytic domains and do not contain substrate-binding domains. We have determined the crystal structure of a family 10 xylanase containing the xylan-binding domain (XBD) from Streptomyces olivaceoviridis E-86 at 1.9 A resolution. The catalytic domain comprises a (beta/alpha)(8)-barrel topologically identical to other family 10 xylanases. XBD has three similar subdomains, as suggested from a triple-repeat sequence, which are assembled against one another around a pseudo-3-fold axis, forming a galactose-binding lectin fold similar to ricin B-chain. The Gly/Pro-rich linker region connecting the catalytic domain and XBD is not visible in the electron density map, probably because of its flexibility. The interface of the two domains in the crystal is hydrophilic, where five direct hydrogen bonds and water-mediated hydrogen bonds exist. The sugar-binding residues seen in ricin/lactose complex are spatially conserved among the three subdomains in XBD, suggesting that all of the subdomains in XBD have the capacity to bind sugars. The flexible linker region enables the two domains to move independently and may provide a triple chance of substrate capturing and catalysis. The structure reported here represents an example where the metabolic enzyme uses a ricin-type lectin motif for capturing the insoluble substrate and promoting catalysis.

Crystallization and preliminary X-ray diffraction studies of aspartic proteinase from Irpex lacteus.

Crystals of ILAP (Irpex lacteus aspartic proteinase) have been obtained by the hanging drop method using ammonium sulfate as a precipitant. The crystals are monoclinic, space group P2(1) with cell dimensions a = 54.5 A, b = 79.6 A, c = 37.5 A, beta = 96.8 degrees. The crystals are quite stable to X-rays and diffract beyond 1.9 A resolution. There is one molecule in the asymmetric unit.

Characterization of a cellulase-free, neutral xylanase from Thermomyces lanuginosus CBS 288.54 and its biobleaching effect on wheat straw pulp.

A xylanase purified from the thermophilic fungus Thermomyces lanuginosus CBS 288.54 was characterized and its potential application in wheat straw pulp biobleaching was evaluated. Xylanase was purified 33.6-fold to homogeneity with a recovery yield of 21.5%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of approx. 26.2 kDa. The purified xylanase had a neutral optimum pH ranging from pH 7.0 to pH 7.5, and it was also stable over pH 6.5-10.0. The optimal temperature of the xylanase was 70-75 degrees C and it was stable up to 65 degrees C. The purified xylanase was found to be not glycosylated. The xylanase was highly specific towards xylan, but did not exhibit other enzyme activity. Apparent Km values of the xylanase for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 4.0, 4.7, 2.0 and 23.4 mg ml-1, respectively. The potential application of the xylanase was further evaluated in biobleaching of wheat straw pulp. The brightness of bleached pulps from the xylanase pretreated wheat straw pulp was 1.8-7.79% ISO higher than that of the control, and showed slightly lower tensile index and breaking length than the control. Although chlorine consumption was reduced by 28.3% during bleaching, the xylanase pretreated pulp (15 U g-1 pulp) still maintained its brightness at the control level. Besides, pretreatment of pulp with the xylanase was also effective at an alkaline pH as high as pH 10.0.

Significant enhancement in the binding of p-nitrophenyl-beta-D-xylobioside by the E128H mutant F/10 xylanase from Streptomyces olivaceoviridis E-86.

Mutagenesis studies were carried out to examine the effects of replacement of either the nucleophile Glu-236 or the acid/base Glu-128 residue of the F/10 xylanase by a His residue. To our surprise, the affinity for the p-nitrophenyl-beta-D-xylobioside substrate was increased by 10(3)-fold in the case of the mutant E128H enzyme compared with that of the wild-type F/10 xylanase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native xylanase and a nitrogen in the mutant) and the alpha-carbon was barely changed. Thus, the alteration of the acid/base functionality (Glu-128 to His mutation) provided a significantly favorable interaction within the E128H enzyme/substrate complex in the ground state, accompanying a reduction in the stabilization effect in the transition state.

Novel sugar-binding specificity of the type XIII xylan-binding domain of a family F/10 xylanase from Streptomyces olivaceoviridis E-86.

The type XIII xylan-binding domain (XBD) of a family F/10 xylanase (FXYN) from Streptomyces olivaceoviridis E-86 was found to be structurally similar to the ricin B chain which recognizes the non-reducing end of galactose and specifically binds to galactose containing sugars. The crystal structure of XBD [Fujimoto, Z. et al. (2000) J. Mol. Biol. 300, 575-585] indicated that the whole structure of XBD is very similar to the ricin B chain and the amino acids which form the galactose-binding sites are highly conserved between the XBD and the ricin B chain. However, our investigation of the binding abilities of wt FXYN and its truncated mutants towards xylan demonstrated that the XBD bound xylose-based polysaccharides. Moreover, it was found that the sugar-binding unit of the XBD was a trimer, which was demonstrated in a releasing assay using sugar ranging in size from xylose to xyloheptaose. These results indicated that the binding specificity of the XBD was different from those of the same family lectins such as the ricin B chain. Somewhat surprisingly, it was found that lactose could release the XBD from insoluble xylan to a level half of that observed for xylobiose, indicating that the XBD also possessed the same galactose recognition site as the ricin B chain. It appears that the sugar-binding pocket of the XBD has evolved from the ancient ricin super family lectins to bind additional sugar targets, resulting in the differences observed in the sugar-binding specificities between the lectin group (containing the ricin B chain) and the enzyme group.

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 Km and kcat 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 Km value for PNP-X2 decreased from the 2.0 mM of FXYN to 0.6 mM and the kcat 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 Km value to 0.3 mM and the kcat 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.

Crystallization and preliminary X-ray crystallographic study of Streptomyces olivaceoviridis E-86 beta-xylanase.

beta-Xylanase from Streptomyces olivaceoviridis E-86 has been crystallized by the hanging drop vapor diffusion method from 25% saturated ammonium sulfate and 2% McIlvaine buffer, pH 5.7. The crystals diffract to at least 1.9 A resolution, and belong to space group P2(1)2(1)2(1), with unit-cell dimensions of a=79.6 A, b=95.2 A, and c=140.3 A. There are probably two xylanase molecules (MW=45 K) per asymmetric unit.

Induction and enhancement of stress proteins in a trichloroethylene-degrading methanotrophic bacterium, Methylocystis sp. M.

The responses of the trichloroethylene-degrading bacterium Methylocystis sp. M to six different water-pollutants, carbon starvation, and temperature-shock (heat and cold) were examined using 2-dimensional gel electrophoresis. Twenty-eight polypeptides were induced, and these stress-induced proteins were classified into three groups. Some of the chemically induced proteins were the same as those induced by carbon starvation and temperature-shock. Two of the polypeptides were induced by trichloroethylene. Trichloroethylene-stress protein synthesis required 1-2 h at a concentration of trichloroethylene that had no effect on growth. Furthermore, 25 stress-enhanced polypeptides were observed, and one of these was enhanced by trichloroethylene. Based on these results, we discuss applications of chemical-stress induction of proteins to establish effective bioremediation and bioassay by methanotrophs.

Synthesis of regioisomeric methyl alpha-L-arabinofuranobiosides.

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

Some properties and action mode of (1-->4)-alpha-L-guluronan lyase from Enterobacter cloacae M-1.

An intracellular alginate lyase was purified from Enterobacter cloacae M-1 by successive fractionation on Q Sepharose FF, SP Sepharose FF, and Sephacryl S-200 HR. The purified enzyme gave a single band on SDS-PAGE and isoelectric focusing. The enzyme easily degraded polyguluronate and produced unsaturated oligoguluronic acids with a wide range of dp. The major end product of the enzyme reaction on polyguluronate was unsaturated triuronic acid. The pattern of oligoguluronic acids (dp 2-9) generated with the enzyme was investigated by fluorophore-assisted carbohydrate electrophoresis. The enzyme was not capable of degrading oligoguluronic acids having dp < 4. The degradation rate of heptaguluronic acid by this enzyme remarkably increased, compared with that of hexaguluronic acid, and heptaguluronic acid had a single preferential point of cleavage by this enzyme. On the basis of the cleavage pattern of oligoguluronic acids, the number of subsites was estimated to be seven for this enzyme. The catalytic site of the enzyme is located between the second and the third subsites from the non-reducing end.

Substrate specificity of alpha-L-arabinofuranosidase from Bacillus subtilis 3-6 toward arabinofurano-oligosaccharides.

The substrate specificity of alpha-L-arabinofuranosidase (EC 3.2.1.55) from Bacillus subtilis 3-6 was explored using methyl 2-O-, methyl 3-O-, and methyl 5-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranosides, and methyl 3,5-di-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside. The enzyme hydrolyzed the methyl alpha-L-arabinofuranobiosides to arabinose and methyl alpha-L-arabinofuranoside in the order of (1-->2)- > (1-->3)- > (1-->5)-linkages. The enzyme hydrolyzed the (1-->3)-linkage more than the (1-->5)-linkage of the methyl alpha-L-arabinofuranotrioside.

Stereoselective synthesis of regioisomers of aldobiouronic acid.

alpha-Glucuronidase is a very important enzyme for the complete hydrolysis of plant hemicellulose, but the substrate specificity of the enzyme has not previously been reported. In this connection, the three regioisomers of O-(alpha-D-glucopyranosyluronic acid)-D-xylose (aldobiouronic acid), 2-O-(alpha-D-glucopyranosyluronic acid)-D-xylose (13), 3-O-(alpha-D-glucopyranosyluronic acid)-D-xylose (14), and 4-O-(alpha-D-glucopyranosyluronic acid)-D-xylose (15), were stereoselectively synthesized to clarify the substrate specificity.

Purification and some properties of intracellular alpha-L-arabinofuranosidase from Aspergillus niger 5-16.

alpha-L-Arabinofuranosidase was purified from a cell-free extract of Aspergillus niger 5-16 by chromatographies on DEAE-Toyopearl, SP-Toyopearl, Ultro-gel AcA 44, Mono P, and TSK-Gel G3000SW. The final preparation thus obtained showed a single band on SDS-polyacrylamide gel electrophoresis. The molecular weight and isoelectric point were 67,000 by SDS-polyacrylamide gel electrophoresis and pH 3.5 by isoelectric focusing. The alpha-L-arabinofuranosidase contained amino acids in the order of Asx > Gly > Ala > Thr > Glx = Ser. The enzyme had maximum activity at pH 4.0 and 60 degrees C, and was stable from pH 4 to 7 and at temperatures up to 30 degrees C. The enzyme activity was not affected considerably by either metal ions or chemical reagents. The enzyme released arabinose from p-nitrophenyl-alpha-L-arabinofuranoside, O-alpha-L-arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D- xylopyranose, and arabinan, but not from O-beta-D-xylopyranosyl-(1-->4)-O-[alpha-L-arabinofuranosyl- (1-->3)]-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose, O-beta-D-xylopyranosyl-(1-->2)-O-alpha-L-arabinofuranosyl-(1-->3)-O-beta -D- xylopyranosyl-(1-->4)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose, gum arabic, or arabinoxylan. The limit of hydrolysis of arabinan was about 58% even when the enzyme was sufficiently in excess.

Purification and some properties of alpha-L-arabinofuranosidase from Bacillus subtilis 3-6.

alpha-L-Arabinofuranosidase (EC 3.2.1.55) was purified from culture supernatant of Bacillus subtilis 3-6. The enzyme had a molecular weight of 61,000 and displayed maximum activity at pH 7.0 and 60 degrees C. It released arabinose from O-alpha-L-arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-x ylopyranos e (A1X2), O-beta-D-xylopyranosyl-(1-->4)-[O-alpha-L-arabinofuranosyl-(1-->3)]- O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (A1X3), and arabinan, but not from O-beta-D-xylopyranosyl-(1-->2)-O-alpha-L- arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-O-beta-D-xylopyr anosyl- (1-->4)-D-xylopyranose (A1X4), arabinoxylan, gum arabic, or arabinogalactan.

Effects of Unsaturated Uronic Acid Residues at Non-reducing End on Bond Cleavage Frequency of Poly(1,4-α-L-guluronide) Lyase from Enterobacter cloacae M-1.

The mode of action of poly(1,4-α-L-guluronide) lyase from Enterobacter cloacae M-1 on unsaturated oligoguluronic acids was studied using fluorophore-assisted carbohydrate electrophoresis. The polyguluronate lyase degraded unsaturated penta-, hexa-, and heptaguluronic acids, but not unsaturated oligoguluronic acids with DPs less than 4. On comparison with the aspect of enzymatic degradation of unsaturated oligoguluronic acid and saturated oligoguluronic acid having the same DP, the former was degraded faster than the latter, and also the cleavage pattern of the polyguluronate lyase on unsaturated oligoguluronic acids was considerably different from that on saturated oligoguluronic acids. From the results described above, we suggest that the affinity of the first subsite from the non-reducing end side of the enzyme to Δ residues is lower than that to GulA residues.

Structure of hardwood xylan and specificity of Streptomyces beta-xylanase toward the xylan.

Three kinds of xylo-oligosaccharides having structures of 3(2)-beta-xylosylxylobiose, 3(2)-beta-xylobiosylxylobiose, and 2(2)-beta-xylobiosyl-xylobiose were isolated from an enzymatic hydrolysate of hardwood xylan with Streptomyces beta-xylanase. The structures suggest that the hardwood xylan has both (1-->2)- and (1-->3)-beta-D-xylopyranosyl linkages in the structure, and the specificity of Streptomyces beta-xylanase toward the stubs is similar to that toward glucuronic acid stubs, but is somewhat different from that toward arabinose and xylosylarabinose stubs.

Nucleotide sequence of alpha-galactosidase cDNA from Mortierella vinacea.

To analyze the primary structure of Mortierella vinacea alpha-galactosidase, a cDNA library of M. vinacea mRNA in lambda gt10 was constructed. A clone, which has an insert size of about 1.4 kilobase pairs, was found to contain the coding region of the mature enzyme. The deduced amino acid sequence share that the mature enzyme consisted of 397 amino acid residues with a molecular mass of 44,350 Da. The sequence identity of the mature enzyme with alpha-galactosidases from Saccharomyces carlsbergensis, Cyamopsis tetragonoloba (guar), and human were 47%, 43%, and 34%, respectively.

Substrate specificity of alpha-glucuronidase isolated from snail acetone powder.

The substrate specificity of a p-nitrophenyl alpha-D-glucopyranosyl-uronic acid-hydrolyzing enzyme (PNP-GAase) isolated from snail acetone powder has been investigated with various substrates, such as p-nitrophenyl alpha-D-glucopyranosyluronic acid (PNP-GA), 2-O-alpha- D-glucopyranosyluronic acid-D-xylose (GA-2X), 2-O-(4-O-methyl-alpha-D-glucopyranosyluronic acid)-D-xylose (MeGA-2X), and O-alpha-D-glucopyranosyluronic acid-alpha-D-glucopyranosiduronic acid (GA-GA). The Km (mM) and Vmax (mumol of glucuronic acid formed/mg of enzyme protein/min) toward these substrates were as follows; 0.13 and 3.21 for PNP-GA, 0.33 and 0.089 for GA-2X, 17.6 and 0.094 for MeGA-2X, and 0.36 and 0.015 for GA-GA, respectively. The results indicate that the PNP-GAase specifically hydrolyzed PNP-GA, however, the enzyme had broad substrate specificity.