Functional Sites Analysis For Typical Cellulase Active Site Architecture

Converting cellulose to ethanol through biological fermentation is one of the methods to solve the world’s energy crisis. While due to the low degradation efficiency and high cost of cellulases, the cellulose resource has not been fully utilized at present. Studying the functional sites in cellulase active site architecture will help to deepen our understanding for the mechnism of cellulases and provide insignts into improving the cellulase activity.In this paper, we sdutyed main cellulase families in CAZy database with bioinformatics methods. Analysis indicates that the origin of cellulases is complicated and all cellulase families may originate from at least five different types of structure. Protein contact potential and structure analysis show that cellulases have similar active site architecture clue to convergent evolution. According to the co-crystal structure of enzyme and substrate, we find out those potential amino acid sites consisted in the cellulase active site architecture of five main cellulase families.Based on homology modelling and structure alignment, we calculated the sequence conservation of amino acid sites in the active site architecture of the five main cellulase families. The conserved sites account for about one-third of all architecture sites. Among this conserved sites, tryptophan tyrosine aspartic acid, histidine and asparagine are with very high occurring frequency. Besides, all the five cellulase families have a conserved tryptophan in the-2subsite and a conserved aspartic acid and (or) asparagine in the-1subsite, which indicates that all cellulases have generality in a sense. Molecular dinamics simulation and structure alignment analysis show that conserved sites are also with conserved spatial position.To find the unconserved functional sites in potential cellulase active site architecture, we developed a new althorithm to analysis the coevolution between amino acid sites in a protein sequence. Obtaining results with the same accuracy as previous althorithm, the new althorithm needs much less amount of data, thus it is more suitable for cellulases. With the new althorithm, we find functional units in all five cellulase families which may evolve independently. Evolutionary test incates that there is selection pressure difference between different branches and sites in each cellulase family. Result of branch-site test demonstrates that the cellobiohydrolase foreground branch and the endoglucanase foreground branch in GHF7have different positive seletion sites, which may relate to the function divergency between cellobiohydrolase and endoglucanase.Based on these results, we analysed the function of conserved sites and some unconserved sites in the active site architecture of five cellulase families, which may lay a foundation for further experiment work. Moreover, this set of bioinformatics methods for enzyme active site architecture analysis can be applied to other proteins to analyze the funcitonal sites in architecture.