Crystal structure of the GH-46 subclass III chitosanase from Bacillus circulans MH-K1 in complex with chitotetraose.

BACKGROUND: Chitosanases (EC 3.2.1.132) hydrolyze chitosan which is a polymer of glucosamine (GlcN) linked by ß - 1,4 bonds, and show cleavage specificity against partially acetylated chitosan containing N-acetylglucosamine (GlcNAc) residues. Chitosanases' structural underpinnings for cleavage specificity and the conformational switch from open to closed structures are still a mystery. METHODS: The GH-46 subclass III chitosanase from Bacillus circulans MH-K1 (MH-K1 chitosanase), which also catalyzes the hydrolysis of GlcN-GlcNAc bonds in addition to GlcN-GlcN, has had its chitotetraose [(GlcN)4]-complexed crystal structure solved at 1.35 Å resolution. RESULTS: The MH-K1 chitosanase's (GlcN)4-bound structure has numerous structural similarities to other GH-46 chitosanases in terms of substrate binding and catalytic processes. However, subsite -1, which is absolutely specific for GlcN, seems to characterize the structure of a subclass III chitosanase due to its distinctive length and angle of a flexible loop. According to a comparison of the (GlcN)4-bound and apo-form structures, the particular binding of a GlcN residue at subsite -2 through Asp77 causes the backbone helix to kink, which causes the upper- and lower-domains to approach closely when binding a substrate. CONCLUSIONS: Although GH-46 chitosanases vary in the finer details of the subsites defining cleavage specificity, they share similar structural characteristics in substrate-binding, catalytic processes, and potentially in conformational change. GENERAL SIGNIFICANCE: The precise binding of a GlcN residue to the -2 subsite is essential for the conformational shift that occurs in all GH-46 chitosanases, as shown by the crystal structures of the apo- and substrate-bound forms of MH-K1 chitosanase.

Age-related morphological and functional changes in the small intestine of senescence-accelerated mouse.

The ability of the small intestine to perform various functions, such as digestion/absorption of nutrients, gradually declines with age. However, the mechanism that causes intestinal senescence remains unclear. Therefore, age-related changes in the jejunum and ileum were evaluated using senescence-accelerated mouse (SAM) strains that possess characteristic phenotypes of aging. In particular, to understand how senescence affects the small intestine, we investigated whether age-related changes in the morphology of the intestinal villi and its capability to digest/absorb nutrients are associated with the senescence phenotypes identified in specific SAM strains. Four SAM strains were selected (SAMP1, SAMP6, SAMP10, and SAMR1; of which SAMR1 served as a control of SAMP strain) and age-related changes in the small intestine were evaluated for each strain. A villus morphological analysis, mRNA expression level analysis of the small intestine-specific molecules, and disaccharidase activity measurement were performed. We observed that the mRNA expression levels of the genes involved in the differentiation of intestinal epithelial cells and in the digestion/absorption of nutrients were markedly decreased in all the SAM strains, especially in the SAMP10 strain. Our results revealed that all the SAM strains spontaneously induced senescence of the small intestine, which occurred due to the disorders affecting the differentiation/maturation system of intestinal epithelial cells. In addition, it was evident that senile phenotypes, such as brain dysfunction, enhanced intestinal senescence in the SAMP10 strain. The results of this study suggest that the brain-intestinal nervous system may play role in maintenance of villous morphology and nutrients uptake via the GLP-2 and IGF-2 signaling pathway.