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• Authors: Ryszard Brzezinski and Marie-Eve Lacombe-Harvey
• Responsible Curator: Ryszard Brzezinski

Substrate specificities

Glycoside hydrolases of family 46 are essentially all 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 acted upon 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 in the sequence SSAENSS, while Asp40 (DIGDGRG) is the general base residue [5, 6]. The latter could activate the nucleophilic water molecule with assistance from residue Thr45 (RGYTGGI) [7]. Analysis of sequence alignments as well as crystallographic evidence showed that the same function is played by residues Glu37 (in the sequence NKPEQDD) , Asp55 (DIEDERG) and Thr60 (RGYTIGL) in the chitosanase from Bacillus circulans MH-K1 [8].

Three-dimensional structures

3D structure of the chitosanase from Streptomyces sp. N174

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 of GH46 enzymes 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]. For the chitosanase from Streptomyces sp N174, the mode of binding of a GlcN hexasaccharide was established as being in conformity with a symmetrical 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 general base residue identification

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

First general acid 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. [Yabuki1988]
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.

   [Boucher1992]

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.

   [Mitsutomi1996]

4. Fukamizo T, Honda Y, Goto S, Boucher I, and Brzezinski R. Reaction mechanism of chitosanase from Streptomyces sp. N174. Biochem J 1995 Oct 15; 311 ( Pt 2) 377-83. pmid:7487871. PubMed HubMed [Fukamizo1995]
5. Boucher I, Fukamizo T, Honda Y, Willick GE, Neugebauer WA, and Brzezinski R. 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 Dec 29; 270(52) 31077-82. pmid:8537367. PubMed HubMed [Boucher1995]
6. Marcotte EM, Monzingo AF, Ernst SR, Brzezinski R, and Robertus JD. X-ray structure of an anti-fungal chitosanase from streptomyces N174. Nat Struct Biol 1996 Feb; 3(2) 155-62. pmid:8564542. PubMed HubMed [Marcotte1996]
7. Lacombe-Harvey ME, Fukamizo T, Gagnon J, Ghinet MG, Dennhart N, Letzel T, and Brzezinski R. Accessory active site residues of Streptomyces sp. N174 chitosanase: variations on a common theme in the lysozyme superfamily. FEBS J 2009 Feb; 276(3) 857-69. doi:10.1111/j.1742-4658.2008.06830.x pmid:19143844. PubMed HubMed [Lacombe-Harvey2009]
8. Saito J, Kita A, Higuchi Y, Nagata Y, Ando A, and Miki K. Crystal structure of chitosanase from Bacillus circulans MH-K1 at 1.6-A resolution and its substrate recognition mechanism. J Biol Chem 1999 Oct 22; 274(43) 30818-25. pmid:10521473. PubMed HubMed [Saito1999]
9. Fukamizo T, Amano S, Yamaguchi K, Yoshikawa T, Katsumi T, Saito J, Suzuki M, Miki K, Nagata Y, and Ando A. Bacillus circulans MH-K1 chitosanase: amino acid residues responsible for substrate binding. J Biochem 2005 Nov; 138(5) 563-9. doi:10.1093/jb/mvi156 pmid:16272568. PubMed HubMed [Fukamizo2005]
10. Monzingo AF, Marcotte EM, Hart PJ, and Robertus JD. Chitinases, chitosanases, and lysozymes can be divided into procaryotic and eucaryotic families sharing a conserved core. Nat Struct Biol 1996 Feb; 3(2) 133-40. pmid:8564539. PubMed HubMed [Monzingo1996]
11. Holm L and Sander C. Structural similarity of plant chitinase and lysozymes from animals and phage. An evolutionary connection. FEBS Lett 1994 Feb 28; 340(1-2) 129-32. pmid:8119396. PubMed HubMed [Holm1994]
12. Tremblay H, Yamaguchi T, Fukamizo T, and Brzezinski R. Mechanism of chitosanase-oligosaccharide interaction: subsite structure of Streptomyces sp. N174 chitosanase and the role of Asp57 carboxylate. J Biochem 2001 Nov; 130(5) 679-86. pmid:11686931. PubMed HubMed [Tremblay2001]
13. Katsumi T, Lacombe-Harvey ME, Tremblay H, Brzezinski R, and Fukamizo T. Role of acidic amino acid residues in chitooligosaccharide-binding to Streptomyces sp. N174 chitosanase. Biochem Biophys Res Commun 2005 Dec 30; 338(4) 1839-44. doi:10.1016/j.bbrc.2005.10.157 pmid:16288718. PubMed HubMed [Katsumi2005]
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.

    [Ando1992]