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 β − 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.