Identification Of A Novel Family Of Carbohydrate-binding Modules With Broad Ligand Specificity

A novel carbohydrate-binding modules?CBM?was identified from C-terminus of endoglucanase C5614-1 derived form the uncultured microorganism of content of buffalo rumen in our previous work.CBMC5614-1 composed of 189 amino acids and showed no significant homology to known CBMs.The most important characteristic of CBMC5614-1 had broad polysaccharide specificity.In order to determine the shortest functional region of binding capacity of CBMC5614-1,the deletion derivatives of N terminus or C terminus of CBMC5614-1 were constructed.Binding experiments of deletion mutants showed that the 16?149 amino acid sequence was the shortest binding region in CBMC5614-1.The 134 amino acids of CBMC5614-1 had 15 homologs in protein database of GenBank.Phylogenetic analysis revealed that these 16 homologous polypeptides were divided into two branches.The identities of the amino acids between polypeptides in the same branch were more than 25%,and that of between differant branches were below 20%.Although members between the two branches had the low identities,they remained to being clustered into a large branch by compared with other CBM families?for example CBM37?.This result showed that CBMC5614-1 and 15 homologs could be classified into two subfamilies.Subfamily a contained CBMC5614₁?the candidate member of subfamily a?,ADR64668-1,CAJ19146,ADR64668-2,ADR64664 and AEK98797;Subfamily b included CBMC35-2?the candidate member of subfamily b?,CBMC29-2,CBMC67-1,ADR64666,ADR64663,ABX76045,ADK55024,AAC36862,ABB46200 and ADA62505.The encoding sequences of homologs of CBMC5614-1?C35-2,C29-2,C67-1,CAJ19146,AAC36862,ABB46200 and ADR64668-1?were cloned and expressed in E.coli.The expressed products were purified and binding capability of homologs of CBMC5614-1 was determined.Binding experiments showed that these homologous polypeptides of CBMc5614-1 had the same polysaccharide binding profiles except ABB46200,which indicated these homologous polypeptides were CBMs.ABB46200 had narrower binding specificity and lower binding capability compared with other homologous polypeptides.It was confirmed that the aromatic amino acids play an important role in recongnizing and binding polysaccharides in known CBM fimilies.Alignment analysis of 14 full length homologs indicated that there were 3 conserved aromatic amino acids in all homologous sequences,they were W17,Y48 and HI 17?numbers according to amino acid sequences of CBMC5614-1?.There were 11 conserved aromatic amino acids?F9,W17,Y41,F45,Y48,F53,Y55,W60,F74,F99,Y104?and one conserved heterocyclic amino acid?H117?in subfamily a?numbers according to amino acid sequences of CBMC5614-1?;there were 11 conserved aromatic amino acids?Y7,Y11,W15,F19,F21,Y34,Y45,Y54,Y73,F87,W133,numbers according to amino acids sequences of CBMC35-2?in subfamily b.These conserved aromatic amino acids of CBMC5614-1 and CBMC35.2 were site-directed mutated into alanines by the method of overlap PCR,respectively,then the binding capability of these site-directed mutants to various polysaccharides were determined.Binding to soluble polysaccharides experiments showed that W17A and W60A of mutants of CBMC5614-1 completely lost binding capacity and the binding capacity of Y48A,F45A,F74A and H117A significantly declined;Results of binding to insoluble polysaccharides indicated that the binding capacity of W17A,W60A and Y48A dramatically decreased and that of F45A,F74 and H117A also decreased in some degree.According to results of binding to soluble polysaccharides of mutants of CBM 035-2,W133A almost completely lost binding capacity and the binding capacity of Y11A,W15A and F19A dramatically decreased,however the binding capacity of F21A,Y34A,Y54A and F73A decreased slightly.The binding capacity of mutants of CBMC35-2 had no obvious change to insoluble polysaccharides compared with wild type CBMC35-2.In this work,the homologs of CBMC5614-1 were proved to be CBMs,and three conserved aromatic amino acids?W17,Y48 and H117?in all homologs played important roles in binding to polysaccharides.These results confirmed that CBMC5614-1 and its homologs formed to a novel family of CBM.