Three-dimensional structure and functional studies of neopullulanase from Thermus brockianus

The structural guided-rational design has been suggested to meet the demand for pullulanase in starch industries. Neopullulanase (EC 3.2.1.135), a pullulan hydrolase type I belonging to the alpha-amylase family that hydrolysis α -1,4-glucosidic bonds in pullulan to produce panose is one such enzyme that can be manipulated. Therefore, this study aimed at modeling three 3- dimensional structures of neopullulanase from Thermus brockianus, a thermophilic bacteria belonging to the thermus genus. The multiple sequence alignment was carried out with Clustal Omega while the domain and 3D structure of this enzyme was predicted with Pfam and four different modeling servers. Model validation was done using SAVES 06 based on the Ramachandran plot and VERIFY3D analysis. YASARA was employed to minimize the energy of the model. PyMOL software was employed to visualize and superimpose the model with the template. The conserved residues and alpha-amylase domain were identified in this enzyme. The RaptorX model was found to be the best with 93.1% residues in the most favoured region based on the Ramachandran plot. The model energy was reduced to -281707.5 kJ/mol with 95.03% of the residues having an average score of 3D-1D score >= 0.2 in the VERIFY3D analysis. TIM barrel active site fold and the three important catalytic residues; Asp329, Glu360 and Asp431 were also identified. This structure prediction will be helpful in the improvement of neopullulanase for large-scale industrial use