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

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.

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 |
6. Marcotte EM, Monzingo AF, Ernst SR, Brzezinski R, and Robertus JD. (1996). X-ray structure of an anti-fungal chitosanase from streptomyces N174. Nat Struct Biol. 1996;3(2):155-62. DOI:10.1038/nsb0296-155 |
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 |
9. Fukamizo T, Amano S, Yamaguchi K, Yoshikawa T, Katsumi T, Saito J, Suzuki M, Miki K, Nagata Y, and Ando A. (2005). Bacillus circulans MH-K1 chitosanase: amino acid residues responsible for substrate binding. J Biochem. 2005;138(5):563-9. DOI:10.1093/jb/mvi156 |
10. Monzingo AF, Marcotte EM, Hart PJ, and Robertus JD. (1996). Chitinases, chitosanases, and lysozymes can be divided into procaryotic and eucaryotic families sharing a conserved core. Nat Struct Biol. 1996;3(2):133-40. DOI:10.1038/nsb0296-133 |
11. Holm L and Sander C. (1994). Structural similarity of plant chitinase and lysozymes from animals and phage. An evolutionary connection. FEBS Lett. 1994;340(1-2):129-32. DOI:10.1016/0014-5793(94)80187-8 |
12. Tremblay H, Yamaguchi T, Fukamizo T, and Brzezinski R. (2001). Mechanism of chitosanase-oligosaccharide interaction: subsite structure of Streptomyces sp. N174 chitosanase and the role of Asp57 carboxylate. J Biochem. 2001;130(5):679-86. DOI:10.1093/oxfordjournals.jbchem.a003034 |
13. Katsumi T, Lacombe-Harvey ME, Tremblay H, Brzezinski R, and Fukamizo T. (2005). Role of acidic amino acid residues in chitooligosaccharide-binding to Streptomyces sp. N174 chitosanase. Biochem Biophys Res Commun. 2005;338(4):1839-44. DOI:10.1016/j.bbrc.2005.10.157 |

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.