Structure and Sequence Based Analysis of Pullulanases: Understanding Dual Catalytic Mechanism.

Background: Starch processing requires a combination of enzymes with other chemical and physical processes, which increases cost and time. Enzymes used in these processes have a characteristic (α/β)8 barrel domain architecture, although, show variable activity. Pullulanase type 1 and isoamylase act on α-1-6 linkage, amylase on α-1-4 linkage whereas pullulanase type 2 acts on both α-1-6, and α-1-4 linkages of starch.

Objective: This article focusses on elucidating the importance of sequence and structural-based differences in pullulanase, that may lead to its dual catalytic nature.

Methods: Initially, sequences and structures of pullulanase type 1, pullulanase type 2, amylase and isoamylase were retrieved from the database (NCBI and PDB). Homology modelling using SWISS-MODEL and PHYRE2 was carried out for predicting the structure of the enzymes with unavailable structures. Further, the modelled structures were validated using ANOLEA, Verify 3D and PROCHECK, structures with high confidence value were selected and used for analysis. Finally, the selected structures were compared by using PDBefold, and their domain alignment and analysis was performed manually using Pymol.

Results: Modelled structures of pullulanase and isoamylase were validated and selected based on the confidence score. Comparative analysis of complete structures low similarity between the enzymes, although, domain analysis showed good similarity. Moreover, alignment of catalytic site residues showed high similarities with the change in orientation of critical site residues (HIS 242, ASP 347 and GLN 375).

Conclusion: The change in orientation of active site residues along with the absence or presence of few residues might play a crucial role in imparting dual functionality.