Research On The Relationship Between Structure And Function Of β-agarase A

Research on glycoside hydrolase is one of the focuses in the glycobiology. Agarases degrade agar or agarose and are classified into two groups according to the modes of action:αandβ-agarases,which hydrolyzeα-1,3 linkages andβ-1,4 linkages in agarose,respectively.Agarases have broad applications in many areas such as food,cosmetic,and medical industries.Therefore,the study of agarases has great theoretical and practical significance.Previously,a new extracellularβ-agarase A (β-AgaA) was founded from marine Pseudoalteromonas sp.CY24.It belongs to the glycoside hydrolases 16 family(GH16).β-AgaA hydrolyzes agarose and agar to generate neoagarotetraose,neoagarohexaose and less amount ofneoagaobiose as final products.It takes neoagarodecaose as the smallest substrate and has two degradation patterns.According to the main hydrolysis products and the hydrolysis modes of degradating neoagarodecaose,β-AgaA shows no obvious similarities with any of the previously defined GH16.In order to reveal the relationship between the structure and function ofβ-AgaA,sequence search,homology modeling and site-directed mutagenesis were used to decipher the key sites of this enzyme.In this paper,bioinformatics methods were used to analyze the commonness and speciality ofβ-agarase in GH16.All of the sequences of agarases were obtained from GenBank database.Sequence similarity was firstly analyzed with ClustalW and BioEdit software and the result indicated the homology of all agarases of GH16 was very low.Next,Espript and HCA were performed with 104Y as the model to analyze the similarity of secondary structure and hydrophobic cluster and the result showed high homology,which indicated that agarases of GH16 might have the similar model. Based on the known crystal structure of agarase 104Y and 104Z,3D structure ofβ-AgaA was predicted with SWISS-Model and SYBYL-FUGUE.The matching information between enzyme and substrate was given by AutoDock and Ligplot analysis.Based on the information given by bioinformatics methods,we speculated that these sites such as H71,F185,N103,S182,TH189-190 and VVTS109-112 have effects on the enzyme activity and catalytic mechanism ofβ-AgaA.Amino acid mutational experiments were carried out based on the results of the bioinformatics analysis.According to the instruction of Quikchange Site-directed Mutagenesis Kit and mega PCR method,using plasmid agaA as the template,the residues on expected sites were changed individually,altogether six mutants:H71N, F185L,N103T,S182I,TH189-190AA and VVTS109-112GCHL.They mutants enzyme were expressed and purified to measure their enzyme activities and degradation patterns of neoagarodecaose.The results showed that the TH189-190AA and VVTS109-112GCHL mutants almost lost all of the enzyme activity.While,the F185L lost part of activity and the activity of the other three mutants were almost the same as that of the wildβ-AgaA.Based on the structure modeling and Ligplot analyzing,the amino acid H71 was founded to locate in the substrate-binding site -4. It was hypothesized to be no effect on the substrate-binding ability because the ability of hydrogen bond interaction was infirm.Wherease,the TH189-190 and WTS109-112 were predicted to locate in the substrate-binding catalytic cavity according to the bioinformatics results.The changes in amino acid side chain and hydrophobic affinity ofβ-AgaA were hypothesized to inhibit the interaction between the enzyme and substrate,resulting in the loss of the enzyme activity.The hydrolysis products of mutants were analyzed with the FACE assay,the results showed that the main products,neoagarotetraose and neoagarohexaose,were the same as that of the wildβ-AgaA.Thus,we concluded that the change of these sites couldn't alter the structure of substrate-binding catalytic cavity,resulting in no effect on the catalytic mechanism.To summarize,bioinformatics methods and site-directed mutagenesis were used to study the relationship between the structure and function ofβ-AgaA,revealing that the effect of the matching information of enzyme-substrate interaction on enzyme activity and degradation pattern.Moreover,this study will promote the research on strucrural and functional relationgship of agarases and other glycoside hydrolases.