Glycoside Hydrolase Family 46

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• Author: ^^^Ryszard Brzezinski^^^
• Responsible Curator: ^^^Ryszard Brzezinski^^^

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Glycoside Hydrolase Family GHnn
Clan	GH-I
Mechanism	inverting
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH46.html

Substrate specificities

Glycoside hydrolases of family 46 are essentially endo-beta-1,4-chitosanases (EC 3.2.1.132) that hydrolyze various links in chitosan, a polymer of beta-1,4-linked D-glucosamine (GlcN) units with a variable content (mostly 0 - 35%) of N-acetyl-D-glucosamine (GlcNAc) [1, 2]. Among the four types of links occurring between these two kinds of subunits in chitosan, all the enzymes examined for their cleavage specificity recognized productively the GlcN-GlcN links. In addition, the chitosanase from Bacillus circulans MH-K1 recognized also GlcN-GlcNAc links [3], while the chitosanase from Streptomyces sp. N174 recognized the GlcNAc-GlcN links [4].

Kinetics and Mechanism

Family GH46 enzymes utilize an inverting mechanism, as shown by NMR [4].

Catalytic Residues

The catalytic residues have been identified by site-directed mutagenesis and crystallography in the chitosanase from Streptomyces sp. N174. The general acid residue is Glu22, while Asp40 is the general base residue [5, 6]. The latter could activate the nucleophilic water molecule with assistance from residue Thr45 [7]. Analysis of sequence alignments as well as crystallographic evidence showed that the same function is played by residues Glu37, Asp55 and Thr60 in the chitosanase from Bacillus circulans MH-K1 [8].

Three-dimensional structures

Two structures have been solved using X-ray crystallography, for the chitosanases from Streptomyces sp. N174 [6] and from Bacillus circulans MH-K1 (wild-type enzyme [8] and mutant K218P [9]. These enzymes have essentially an alpha-helical fold, with two globular domains separated by the active site cleft for substrate binding. The cleft is bordered on the upper face by a three-stranded beta-sheet. The structure is similar to the 3D fold of the well studied lysozyme of bacteriophage T4 of Escherichia coli belonging to family GH24 [6] and, to some extent, to the structures of lysozymes from families GH22, GH23 as well the chitinases from family GH19 [10]. These five families are sometimes grouped in the "lysozyme superfamily" [7, 11]. The crystal structures, completed by site-directed mutagenesis have also revealed several residues involved in substrate binding [6, 9, 12, 13]. While a 4+2 model of substrate binding has been initially proposed for a GlcN hexasaccharide [6], the mode of binding was later established as being in conformity with a 3+3 model, based on the analysis of products of hydrolysis [12].

Family Firsts

First primary sequence determination

Chitosanase from Bacillus circulans MH-K1 [14].

First sterochemistry determination

Chitosanase from Streptomyces sp. N174 by NMR [4].

First catalytic nucleophile identification

Chitosanase from Streptomyces sp. N174 by sequence conservation and mutagenesis [5] and by X-ray crystallography [6].

First general acid/base residue identification

Chitosanase from Streptomyces sp. N174 by sequence conservation and mutagenesis [5] and by X-ray crystallography [6].

First 3-D structure

Chitosanase from Streptomyces sp. N174 by X-ray crystallography [6].

References

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1. Yabuki, M., Uchiyama, A., Suzuki, K., Ando, A., Fujii, T. (1988) Purification and properties of chitosanase from Bacillus circulans MH-K1. Journal of General and Applied Microbiology 34:255-270.

2. Boucher, I., Dupuy, A., Vidal, P., Neugebauer, WA., Brzezinski, R. (1992) Purification and characterization of a chitosanase from Streptomyces N174. Applied Microbiology and Biotechnology 38:188-193.

3. Mitsutomi, M., Ueda, M., Arai, M., Ando, A., Watanabe, T. (1996) Action patterns of microbial chitinases and chitosanases on partially N-acetylated chitosan. Chitin Enzymology, vol. 2, pp 273-284.

14. Ando, A., Noguchi, K., Yanagi, M., Shinoyama, H., Kagawa, Y., Hirata, H., Yabuki, M., Fujii, T. (1992) Primary structure of chitosanase produced by Bacillus circulans MH-K1. Journal of General and Applied Microbiology 38:135-144.