Chitosanase families




THE FAMILY GH80


The family 80 of glycoside hydrolases includes chitosanases that are exclusively of bacterial origin. So far (2013), this family contains 15 proteins and the chitosanase activity has been demonstrated experimentally for four of them, namely that from Matsuebacter chitosanotabidus 3001 (20), Sphingobacterium multivorum (48), Proteobacterium KMU3 and Mitsuaria sp. 141(114). Chitosanase of Matsuebacter chitosanotabidus 3001, named ChoA, is the most extensively studied protein among members of the GH80 family (20, 62). Directed mutagenesis studies suggested that ChoA uses two glutamates as catalytic residues which differs from the aspartate / glutamate duo usually observed in chitosanases or chitinases. The same study also identified several other residues important for ChoA activity. The authors suggested that an arginine located near the catalytic residues plays a role in controlling the pKa of the catalytic residues or is involved in maintaining the structure of the enzyme. According Shimono et al. (62), the presence of six cysteine residues in ChoA seems essential to its activity. Formation of a disulfide bridge between two of the six cysteines has also been demonstrated in this study. In 2005, Yun et al. (62) isolated and identified eleven chitosanases belonging to GH80. Site-directed mutagenesis performed on the chitosanase from Mitsuaria sp. 141 revealed that three non-conserved residues are important for maintaining the enzyme activity and also contribute to the high optimum temperature of this chitosanase (65 Celsius) 114).
An alignment analysis performed in our laboratory has shown that, most probably, GH 80 family enzymes share a common, highly conserved "active site" module with the enzymes belonging to Family GH46 (56).


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

T-Coffee (100) alignment of chitosanase sequences:
* - residue conserved in all sequences; arrows show catalytic residues. Residues included in GH46 and GH80 molecular signature are highlighted in grey. STR_N174, Streptomyces sp. N174; NOC_N106, Nocardioides sp. N106; NOC_K01H, Nocardioides sp. K-01; BAC_CIRC, Bacillus circulans MH-K1; AMY_CS02, Amycolatopsis sp. CsO-2 ; BAC_AMYL, Bacillus amyloliquefaciens ; BAC_EHIM, Paenibacillus ehimensis EAG-1; BAC_KFB, Bacillus sp. KFB-CO4; BAC_SUBT, Bacillus subtilis ; BUR_GLAD, Burkhoderia gladioli CHB101; SAV2015, gene SAV2015 from Streptomyces avermitilis ; SAV6191, gene SAV6191 from Streptomyces avermitilis ; SCO0677, chitosanase CsnA from Streptomyces coelicolor A3(2); PSE_A01, Pseudomonas sp. A-01; SAMBOF, Streptomyces ambofaciens ATCC 23977; CHR_VIOL, Chromobacterium violaceum ATCC 12472; BAC_AMJ1, Bacillus amyloliquefaciens MJ-1; BAC_AMFZ, Bacillus amyloliquefaciens FZB42; PAENIBAC, Paenibacillus sp. BH-2005; MICROBAC , Microbacterium sp. OU01; REN_SALM, Renibacterium salmoninarum ATCC 33209; BAC_DAU, Bacillus sp. DAU101; CLO_BEI, Clostridium beijerinckii NCIMB 8052; CHV_CVK2, Chlorella virus CKV2; CHV_PBU, Paramecium bursaria Chlorella virus 1; MAT_CHIT, Matsuebacter chitosanotabidus 3001; SPH_MULT, Shingobacterium multivorum ; PRO_KNU3, Proteobacterium sp. KNU3; COM_SP46, Comamonas sp. 46; FLA_SP2, Flavobacterium sp. 2; HER_SP27, Herbaspirillum sp. 27; HER_SP9, Herbaspirillum sp. 9; MAT_SP12, Mitsuaria chitosanitabida 12; MAT_SP13, Mitsuaria chitosanitabida 13; MAT_SP67, Mitsuaria chitosanitabida 67; SPH_SP62, Shingobacterium sp.62; STE_SP22, Stenotrophomonas sp. 22; STE_SP45, Stenotrophomonas sp. 45.

In 2002, Shimono and coworkers (62) proposed another alignment for this region from some chitosanases belonging to families 46 and 80:


MAT_CHIT     119 YPENGTTNYQEVGPWRYCEVDYEAAQGISDYRGDTFGPVGVTTV-GD 164
STR_N174      60 SAENSSLDWKAQ--YKYIED-------IGDGRGYTGGIIGFCSGTGD  97
NOC_N106      61 SAENSSLDWKSQ--YKYIED-------IKDGRGYTAGIIGFCSGTGD  98
BAC_CIRC      77 KPEQDDLNWIKY--YGYCED-------IEDERGYTIGLFGATTG-GS 113
PBCV-1       105 LPENSTTQWWKN--YNYASC-------LKDGRGWTVTIYGACSGTGR 142
                   E             Y            D RG T        G

This alignment highlights an important difference between family 80 and 46 discovered by Shimono et al (62): the best candidate for a catalytic residue in the chitosanase from Matsuebacter chitosanotabidus is Glu141, an amino acid that does not seem to align with the catalytic residue from family 46 chitosanases (Asp80 in Streptomyces sp. N174). Without any evidence from crystallography, it is difficult, at present, to decide which alignment is a better representation of chitosanase sequence relationships. Nevertheless, since the nature of the catalytic amino acids is a characteristic maintained within the same family of glycoside hydrolases, the classification of chitosanases families GH 46 and GH 80 in two distinct families appears justified.




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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