Sachio Goto

Specificity of chitosanase from Bacillus pumilus

Partially (25-35%) N-acetylated chitosan was digested by chitosanase from Bacillus pumilus BN-262, and structures of the products, partially N-acetylated chitooligosaccharides, were analyzed in order to investigate the specificity of the chitosanase. The chitosanase produced glucosamine (GlcN) oligosaccharides abundantly, indicating that the chitosanase splits the beta-1,4-glycosidic linkage of GlcN-GlcN. The chitosanase also produced hetero-oligosaccharides consisting of glucosamine and N-acetyl-D-glucosamine (GlcNAc). Three types of the hetero-oligosaccharides purified by cation-exchange chromatography and HPLC were found to have GlcNAc residue at their reducing end and GlcN residue at their non-reducing end, indicating that the chitosanase can also split the linkage of GlcNAc-GlcN. The determination of the mode of action toward partially N-acetylated chitosan enables a classification of chitosanases according to their specificities and a more precise definition of chitosanases.

Substrate size dependence of lysozyme-catalyzed reaction

In the study of the mechanism of lysozyme-catalyzed reactions, it has been assumed that the rate constants in the catalytic process, the catalytic activity of catalytic group Glu 35, are independent of the degree of polymerization (size) of the substrate. The characteristics of substrate binding subsite F have recently been reexamined and the substrate binding mode at this subsite has been demonstrated to be more complex than expected from a model based on an X-ray analysis of the lysozyme-substrate complex. In the present study, the time courses of the lysozyme-catalyzed reactions with the substrates chitotetraose [(GlcNAc)4], chitopentaose [(GlcNAc)5], and chitohexaose [(GlcNAc)6], of 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc), were obtained experimentally with high-performance liquid chromatography. From the experimental time courses, the values of the rate constants, k+1 (the cleavage of glycosidic linkage) and k-1/k+2 (relative efficiency of transglycosylation), were obtained by a data-fitting method with computer simulation of the lysozyme-catalyzed reaction (A. Masaki et al. (1981) J. Biochem. 90, 1167-1175). As a result, it was found that the k+1 value is dependent on the substrate size and the value of the binding free energy of subsite F is considerably smaller than previously estimated. The substrate size dependence of the k+1 value is considered to relate closely to the fine structure of the binding and catalytic sites.

Retention of anomeric form in lysozyme-catalyzed reaction.

A lysozyme-catalyzed reaction is initiated by a cleavage of the beta-1, 4-glucosaminide linkage, followed by hydration and transglycosylation. Since all glycosides produced by transglycosylation have beta-glycosidic linkages between the sugar and the acceptor moieties, the lysozyme-catalyzed reaction has been classified as an anomer-retention reaction. However, there is no experimental evidence on the anomer retention of the new reducing residue produced by the hydrolysis of the substrate. In the present study, an attempt was made to determine the anomeric form of the GlcNAc residue at the reducing end in nascent hydrolytic products. The anomeric forms of the enzymatic products were separated and quantitatively analyzed by high-performance liquid chromatography. The amounts of alpha- and beta-anomers in the product were plotted against the reaction time. Computer analysis of the experimental data indicated that the nascent hydrolytic product takes only the beta-anomeric form and that the alpha-anomer is formed from beta-anomer by mutarotation.

Thermal unfolding of chitosanase from Streptomyces sp. N174: role of tryptophan residues in the protein structure stabilization

Tryptophan residues in chitosanase from Streptomyces sp. N174 (Trp28, Trp101, and Trp227) were mutated to phenylalanine, and thermal unfolding experiments of the proteins were done in order to investigate the role of tryptophan residues in thermal stability. Four types of mutants (W28F, W101F, W227F and W28F/W101F) were produced in sufficient quantity in our expression system using Streptomyces lividans TK24. Each unfolding curve obtained by CD at 222 nm did not exhibit a two-state transition profile, but exhibited a biphasic profile: a first cooperative phase and a second phase that is less cooperative. The single tryptophan mutation decreased the midpoint temperature (Tm) of the first transition phase by about 7 degrees C, and the double mutation by about 11 degrees C. The second transition phase in each mutant chitosanase was more distinct and extended than that in the wild-type. On the other hand, each unfolding curve obtained by tryptophan fluorescence exhibited a typical two-state profile and agreed with the first phase of transition curves obtained by CD. Differential scanning calorimetry profiles of the proteins were consistent with the data obtained by CD. These data suggested that the mutation of individual tryptophan residues would partly collapse the side chain interactions, consequently decreasing Tm and enhancing the formation of a molten globule-like intermediate in the thermal unfolding process. The tryptophan side chains are most likely to play important roles in cooperative stabilization of the protein.

Integration of Chitin-Degrading Microbes into Biological Control System for Fusarium Wilt of Strawberry

It has been known that an amendment of chitin to soil leads to an increase in the population of chitinolytic microbes and a decrease in the population of soilborne fungal plant pathogens (Boller, 1986; Mitchell and Alexander, 1962). This phenomenon has been applied with some successes to microbial control disease (Sneh et al., 1971; Sneh, 1981). In combination with a chitin-degrading microorganisms (Streptomyces sp.)and a root-colonizing antagonistic bacterium (Serratia marcescens), we successfully protected tomato plants from Fusarium wilt (Toyoda et al., submitted). In our system, an amendment of chitin to soils was essential for promoting preferential multiplication of chitin-degrading microorganisms and subsequent growth of antagonistic rhizoplane bacteria. These previous studies suggested that the pathogen could be effectively suppressed by the use of chitin-degrading microorganisms with antifungal activities, and that this system sould be practically used for the reduction of inoculum potential of pathogens in infested soils, provided the growth of these antagonists was promoted preferentially.

Novel Trimeric Adenylate Kinase from an Extremely Thermoacidophilic Archaeon, Sulfolobus solfataricus: Molecular Cloning, Nucleotide Sequencing, Expression in Escherichia coli,…

A gene coding for adenylate kinase was cloned from an extremely thermoacidophilic archaeon Sulfolobus solfataricus. The open reading frame of the sequenced gene consisted of 585 nucleotides coding for a polypeptide of 195 amino acid residues with a calculated molecular weight of 21,325. Although the S. solfataricus adenylate kinase, which belonged to the small variants of the adenylate kinase family, had low sequence identities with bacterial and eukaryotic enzymes, a functionally important glycine-rich region and also two invariant arginine residues were conserved in the sequence of the S. solfataricus enzyme. The recombinant enzyme, overexpressed in Escherichia coli and purified to homogeneity, had high affinity for AMP and high thermal stability, comparable to the extremely thermostable enzyme from a similar archaeon, S. acidocaldarius. Furthermore, gel filtration and sedimentation analyses showed that the S. solfataricus adenylate kinase was a homotrimer in solution, which is a novel subunit structure for nucleoside monophosphate kinases.

Hen-egg-white lysozyme modified with histamine. State of the imidazolylethyl group covalently attached to the binding site and its effect on the sugar-binding ability.

The chemical modification of Asp101 which is located at the upper end-most site (site A) of the binding cleft of hen egg white lysozyme affects the sugar residue binding of the midmost site (site C) in addition to that of site A, and results in the considerable decrease in the enzymic activity [Fukamizo, T., Hayashi, K. & Goto, S. (1986) Eur. J. Biochem. 158, 463-467]. In the present study, Asp101 was modified with histamine and converted to [2-imidazol-4(5)-ylethyl]asparagine. Contrary to the findings described above, the specific activity of the modified lysozyme was higher than that of the native lysozyme by a factor of about two, and the loss of sugar residue binding ability caused by the modification was found to be restricted to site A. From the H-NMR spectra of the modified lysozyme, the pKa value of the imidazolylethyl group covalently attached to Asp101 was 7.1, and was higher than that of N-acetylhistidinemethylamide (6.65). This indicates that the imidazolylethyl moiety is not exposed to the solvent but adheres to the surface of the lysozyme molecule in an unidentified manner. When N-acetylglucosamine trisaccharide [GlcNAc)3] was added to the modified lysozyme, the 1H-NMR signals of H2 and H4 of the imidazolylethyl group were strongly affected. This indicates that the imidazolylethyl moiety is located near (GlcNAc)3 binding region. When the H gamma signal of Ile98 was saturated, nuclear Overhauser effects were observed on H2 and H4 resonances of the imidazolylethyl moiety. NOE was also observed on the signal of Trp63 H6 upon the saturation of the H4 signal of the imidazolylethyl moiety. Thus, the imidazolylethyl moiety should be located near Trp63 and Ile98, which are in the hydrophobic box most proximal to the sugar binding cleft. This situation of the imidazolylethyl moiety did not result in steric hindrance to the sugar residue binding at sites B and C. The modification affected only the sugar residue binding at site A, and resulted in the enhanced activity.