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. Furthermore, 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

Content is to be added here.

Family Firsts

First sterochemistry determination

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First catalytic nucleophile identification

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First general acid/base residue identification

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First 3-D structure

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References

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, W. A., 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.

4. Fukamizo T, Honda Y, Goto S, Boucher I, and Brzezinski R. (1995). Reaction mechanism of chitosanase from Streptomyces sp. N174. Biochem J. 1995;311 ( Pt 2)(Pt 2):377-83. DOI:10.1042/bj3110377 |
5. Boucher I, Fukamizo T, Honda Y, Willick GE, Neugebauer WA, and Brzezinski R. (1995). Site-directed mutagenesis of evolutionary conserved carboxylic amino acids in the chitosanase from Streptomyces sp. N174 reveals two residues essential for catalysis. J Biol Chem. 1995;270(52):31077-82. DOI:10.1074/jbc.270.52.31077 |
7. Lacombe-Harvey ME, Fukamizo T, Gagnon J, Ghinet MG, Dennhart N, Letzel T, and Brzezinski R. (2009). Accessory active site residues of Streptomyces sp. N174 chitosanase: variations on a common theme in the lysozyme superfamily. FEBS J. 2009;276(3):857-69. DOI:10.1111/j.1742-4658.2008.06830.x |
8. Saito J, Kita A, Higuchi Y, Nagata Y, Ando A, and Miki K. (1999). Crystal structure of chitosanase from Bacillus circulans MH-K1 at 1.6-A resolution and its substrate recognition mechanism. J Biol Chem. 1999;274(43):30818-25. DOI:10.1074/jbc.274.43.30818 |