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To identify the amino acids responsible for the substrate binding of chitosanase from Bacillus circulans MH-K1 (MH-K1 chitosanase), Tyr148 and Lys218 of the chitosanase were mutated to serine and proline, respectively, and the mutated chitosanases were characterized. The enzymatic activities of Y148S and K218P were found to be 12.5% and 0.16% of the wild type, respectively. When the (GlcN)3 binding ability to the chitosanase was evaluated by fluorescence spectroscopy and thermal unfolding experiments, the binding abilities of both mutant enzymes were markedly reduced as compared with the wild type enzyme. The affinity of the enzyme for the trisaccharide decreased by 1.0 kcal/mol of binding free energy for Y148S, and 3.7 kcal/mol for K218P. The crystal structure of K218P revealed that Pro218 forms a cis-peptide bond and that the state of the flexible loop containing the 218th residue is considerably affected by the mutation. Thus, we conclude that the flexible loop containing Lys218 plays an important role in substrate binding, and that the role of Tyr148 is less critical, but still important, due to a stacking interaction or hydrogen bond.

Chitosanase is an enzyme that belongs to the glycoside hydrolase family and is characterized by its ability to catalyze the hydrolytic cleavage of chitosan, a polycationic carbohydrate derived from chitin by partial or complete deacetylation. Chitosan is a mixed polysaccharide containing 1,4 linked residues of D glucosamine (GlcN) and N acetyl D glucosamine (GlcNAc). The differences in the mechanism of chitosan hydrolysis among the various groups of enzymes, such as lysozymes, chitinases, and chitosanases, were examined by experiments that analyzed the structure (i.e., the sequence) of the oligosaccharide products from enzymatic hydrolysis. From these sequences, the cleavage specificity of several enzymes could be deduced. Fukamizo et al . 1) proposed classifying chitosanases as enzymes that hydrolyze chitosan without splitting the linkage GlcNAc GlcNAc. Conversely, chitinases could cleave the GlcNAc GlcNAc linkage, but not the GlcN GlcN linkage. Chitosanases were further subdivided into three classes according to their cleavage specificity; class I enzymes could split both GlcN GlcN and GlcNAc GlcN linkages; class II enzymes could split only GlcN GlcN linkages; and class III enzymes could split both GlcN GlcN and GlcN GlcNAc linkages. The recognition mechanisms of the chitosanases are thus complicated, making it difficult to unequivocally distinguish from the other chitinolytic enzymes. This situation lead us to examine the mode of substrate binding of chitosanases.A number of chitosanases have been isolated from various bacteria and fungi, and their genes have been cloned and sequenced. These chitosanases belong to various GH (glycosyl hydrolase) families, GH 5, GH 8, GH 46, GH 75 and GH 80 according to the amino acid sequences. 2)Among these chitosanases sequenced thus far, the enzymes from Streptomyces sp. N174 (N174 chitosanase) and Bacillus circulans MH K1 (MH K1 chitosanase), belonging to family GH 46, have been most intensively studied based on their X ray crystal structures. 3,4) In this article, we review the recent findings from the substrate binding experiments of family GH 46 chitosanases conducted in our laboratory. N174 Chitosanase.N174 chitosanase is classified under class I chitosanases according to its splitting specificity, 5) and its properties have been reviewed by Fukamizo and Brzezinski. Abstract: Mode of substrate-binding of chitosanases from Streptomyces sp. N174 (N174 chitosanase) and Bacillus circulans MH-K1 (MH-K1 chitosanase) was examined by site-directed mutagenesis and physicochemical techniques, including thermal unfolding, fluorescence spectroscopy, and X-ray crystallography. Asp57 located at the central portion of the binding cleft of N174 chitosanase was mutated to asparagine and alanine (D57N and D57A), and the relative activities of the mutated enzymes were 72 and 0.5% of that of the wild type, respectively. Thermal unfolding experiments in the presence of (GlcN)n clearly indicated the importance of Asp 57 for substrate binding. Kinetic analysis of (GlcN)6 degradation cata...

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