Chitosanase families




THE FAMILY GH46

The most studied chitosanases adhering to the Enzyme Commission definition belong to the family 46 of glycoside hydrolases (see also ref. 11).This family includes (so far) only enzymes with chitosanase activity and has over a hundred members. These enzymes are not only of bacterial origin but also from viruses.
Several three-dimensional structures of GH46 chitosanases have been solved: that of Streptomyces sp. N174 (38) and that of Bacillus circulans MK-1 (49), what significantly improved the understanding of the catalytic mechanism and the interaction with substrate. Meanwhile, many molecular studies identified catalytic residues and other residues involved in substrate binding in several members of this family (36, 49,67, 42, 89). Below we show the alignment of 24 chitosanases belonging to GH 46.

NOTE: Throughout this website, as well as in most of our publications, the numbering of amino acid residues in the N174 chitosanase begins with the first a.a. of the mature, secreted protein. This will differ from the numbering used in databases on the internet where numbering begins usually with the first a.a. of the immature protein. To convert database numbering into our numbering you should simply subtract 40 residues (the length of the signal peptide) from the database number.

T-Coffee (100) alignment of chitosanase sequences:
* - residue conserved in all sequences. Arrows show catalytic residues.

STR_N174, Streptomyces sp. N174 (12); NOC_N106, Nocardioides sp. N106 (13); BAC_CIRC, Bacillus circulans MH-K1(17,49) ; AMY_CS02, Amycolatopsis sp. CsO-2 (GenBank locus AB041775.1); BAC_AMYL, Bacillus amyloliquefaciens (18); BAC_EHIM, Paenibacillus ehimensis EAG-1(19, 43) ; BAC_KFB, Bacillus sp. KFB-CO4 (53) alias Bacillus sp. strain CK4 (55); BAC_SUBT, Bacillus subtilis (16, 60); BUR_GLAD, Burkhoderia gladioli CHB101 (45, 58); SAV2015, SAV2015 gene from Streptomyces avermitilis (90) ; SAV6191, SAV6191 gene from Streptomyces avermitilis (90); SCO0677, SCO0677 gene from Streptomyces coelicolor A3(2) (54) ; PSE_A01, Pseudomonas sp. A-01 (89); SAMBOF , Streptomyces ambofaciens ATCC 23977 (91) ; CHR_VIOL, Chromobacterium violaceum ATCC 12472 (GenBank locus AAQ61593.1); BAC_AMJ1, Bacillus amyloliquefaciens MJ-1(18); BAC_AMFZ, Bacillus amyloliquefaciens FZB42(92); PAENIBAC, Paenibacillus sp. BH-2005 (GenBank locus ABC17783.1); MICROBAC , Microbacterium sp. OU01 (93); REN_SALM, Renibacterium salmoninarum ATCC 33209 (GenBank locus ABY24857.1); BAC_DAU, Bacillus sp. DAU101 (94); CLO_BEI, Clostridium beijerinckii NCIMB (95); CHV_CVK2, Chlorella virus CKV2 (15); CHV_PBU, Paramecium bursaria Chlorella virus 1 (96).


Based on alignments of the primary sequences of chitosanases from GH 46 family, Tremblay et al.(56) found that the N-terminal part of these proteins contains several highly conserved residues. They proposed the following protein motif as the molecular signature of GH 46 chitosanases (PROSITEdatabase (99)).

E-[DNQ]-x(8,17)-Y-x(7)-D-x-[RD]-[GP]-x-[TS]-x(3)-[AIVFLY]-G-x(5,11)-D

According to Tremblay et al., this signature is not only present in GH 46 family chitosanases, but also in those belonging to the GH 80 family (see also Family 80 section). Based on data provided by mutagenesis studies conducted on the chitosanase from Streptomyces sp. N174 (36, 26), this signature includes both catalytic residues, besides other residues involved in stability and substrate binding.

Residues located at the C-terminus are less conserved among members of the GH family 46 than those located at the N-terminus. These differences are reflected by the formation of distinct clusters in the phylogenetic tree of the family.

Unrooted phylogenic tree generated from whole-length sequence alignment of GH 46 chitosanase sequences.
* - residue conserved in all sequences. Arrows show catalytic residues.


STR_N174, Streptomyces sp. N1<74 (12); NOC_N106, Nocardioides sp. N106 (13); BAC_CIRC, Bacillus circulans MH-K1(17,49) ; AMY_CS02, Amycolatopsis sp. CsO-2 (GenBank locus AB041775.1); BAC_AMYL, Bacillus amyloliquefaciens (18); BAC_EHIM, Paenibacillus ehimensis EAG-1(19, 43) ; BAC_KFB, Bacillus sp. KFB-CO4 (53) alias Bacillus sp. strain CK4 (55); BAC_SUBT, Bacillus subtilis (16, 60); BUR_GLAD, Burkhoderia gladioli CHB101 (45, 58); SAV2015, SAV2015 gene from Streptomyces avermitilis (90) ; SAV6191, SAV6191 gene from Streptomyces avermitilis (90); SCO0677, SCO0677 gene from Streptomyces coelicolor A3(2) (54) ; PSE_A01, Pseudomonas sp. A-01 (89); SAMBOF , Streptomyces ambofaciens ATCC 23977 (91) ; CHR_VIOL, Chromobacterium violaceum ATCC 12472 (GenBank locus AAQ61593.1); BAC_AMJ1, Bacillus amyloliquefaciens MJ-1(18); BAC_AMFZ, Bacillus amyloliquefaciens FZB42(92); PAENIBAC, Paenibacillus sp. BH-2005 (GenBank locus ABC17783.1); MICROBAC , Microbacterium sp. OU01 (93); REN_SALM, Renibacterium salmoninarum ATCC 33209 (GenBank locus ABY24857.1); BAC_DAU, Bacillus sp. DAU101 (94); CLO_BEI, Clostridium beijerinckii NCIMB (95); CHV_CVK2, Chlorella virus CKV2 (15); CHV_PBU, Paramecium bursaria Chlorella virus 1 (96); JAN_4239, Janthinobacterium sp. strain 4239(102).

Most of well-characterized chitosanases belong to the same cluster as Streptomyces sp. N174 chitosanase: CsnA (also called ScCsn46A or Sco0677) from Streptomyces ceolicolor A3(2) (97, 98), CsnN106 from Nocardioides sp. N106 (13) (new name: Kitasatospora sp. N106), CtoA from Amycolatopsis sp. CsO-2 (101) and CtoI from Pseudomonas sp. A-01(89).

In 2010, Jonhsen et al. isolated and characterized a novel GH 46 chitosanase from the psychrotrophic Janthinobacterium sp. strain 4239 (102). Although this chitosanase belongs to the same cluster as Streptomyces sp. N174 chitosanase, alignment of the Janthinobacterium chitosanase (jan_4239) with that of Streptomyces sp. N174 revealed the lack of a general base catalytic residue equivalent of the aspartate 40 from Streptomyces sp. N174 chitosanase, which is believed to be present in all other known GH46 chitosanases (102).

str_n174 AGAGLDDPHKKEIAMELVSSAENSSLDWKAQYKYIEDIGDGRGYTGGIIGFC 52 jan_4239 AADGLSAPAKKEIAMQLVSSSENSSLDWKAQYAYIEDIAMG-GYTGGIIGFT 79 *. **. * ******:****:*********** *****. * *********

Bacillus circulans MH-K1 chitosanase (Cho1), which belongs to a different cluster from that of Streptomyces sp. N174, is also well-characterized (103,49,104). Two articles were dedicated to the chitosanase (Csn) from Bacillus subtilis ( (105,106).

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This page was created by Ryszard Brzezinski, Marie-Ève Lacombe-Harvey and Andrzej Neugebauer.
Questions? Proposals? Comments? Write to Ryszard.Brzezinski@USherbrooke.ca
Last updated: November 2013