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Protein & Peptide Letters


ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Research Article

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

Author(s): Shubham Vashishtha, Tushar S. Barwal and Saurabh Bansal*

Volume 26 , Issue 12 , 2019

Page: [893 - 903] Pages: 11

DOI: 10.2174/0929866526666190820160611

Price: $65


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.

Keywords: Starch debranching enzymes, homology modelling, pullulanase type 1, α-Amylase, isoamylase, pullulanase type 2, structural alignment

Graphical Abstract
Hii, S.L.; Tan, J.S.; Ling, T.C.; Ariff, A.B. Pullulanase: role in starch hydrolysis and potential industrial applications. Enzyme Res., 2012, 2012921362
[] [PMID: 22991654]
Schenck, F.W.; Hebeda, R.E. Starch hydrolysis products: worldwide technology, production, and application; VCH Verlag: Deutschland, 1992.
Swinkels, J. Sources of Starch, its Chemistry and Physics. In: Starch Conversion Technology; Van Beynum, G.M.; Roel, J.A., Eds.; Marcel Dekker Inc.: New York, 1985, pp. 15-46.
Nakamura, Y. Some properties of starch debranching enzymes and their possible role in amylopectin biosynthesis. Plant Sci., 1996, 121(1), 1-18.
van der Maarel, M.J.; van der Veen, B.; Uitdehaag, J.C.; Leemhuis, H.; Dijkhuizen, L. Properties and applications of starch-converting enzymes of the alpha-amylase family. J. Biotechnol., 2002, 94(2), 137-155.
[] [PMID: 11796168]
Asha, R.; Niyonzima, F.; Sunil, S. Purification and properties of pullulanase from Bacillus halodurans. Int. Res. J. Biol. Sci., 2013, 2(3), 35-43.
Noorwez, S.; Ezhilvannan, M.; Satyanarayana, T. Production of a high maltose-forming, hyperthermostable and Ca2+-independent amylopullulanase by an extreme thermophile Geobacillus thermoleovorans in submerged fermentation. Indian J. Biotechnol., 2006, 5, 337-345.
Jespersen, H.M.; MacGregor, E.A.; Henrissat, B.; Sierks, M.R.; Svensson, B. Starch- and glycogen-debranching and branching enzymes: prediction of structural features of the catalytic (beta/alpha)8-barrel domain and evolutionary relationship to other amylolytic enzymes. J. Protein Chem., 1993, 12(6), 791-805.
[] [PMID: 8136030]
Bernfeld, P. Amylases, alpha and beta. Methods Enzymol., 1955, 1, 149-158.
Coordinators, N.R. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res., 2017, 45(D1), D12-D17.
[] [PMID: 27899561]
Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol., 1990, 215(3), 403-410.
[] [PMID: 2231712]
Rose, P.W.; Prlić, A.; Altunkaya, A.; Bi, C.; Bradley, A.R.; Christie, C.H.; Costanzo, L.D.; Duarte, J.M.; Dutta, S.; Feng, Z.; Green, R.K.; Goodsell, D.S.; Hudson, B.; Kalro, T.; Lowe, R.; Peisach, E.; Randle, C.; Rose, A.S.; Shao, C.; Tao, Y.P.; Valasatava, Y.; Voigt, M.; Westbrook, J.D.; Woo, J.; Yang, H.; Young, J.Y.; Zardecki, C.; Berman, H.M.; Burley, S.K. The RCSB protein data bank: integrative view of protein, gene and 3D structural information. Nucleic Acids Res., 2017, 45(D1), D271-D281.
[] [PMID: 27794042]
Berman, H.M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.N.; Weissig, H.; Shindyalov, I.N.; Bourne, P.E. The Protein Data Bank. Nucleic Acids Res., 2000, 28(1), 235-242.
[] [PMID: 10592235]
Schwede, T.; Kopp, J.; Guex, N.; Peitsch, M.C. SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res., 2003, 31(13), 3381-3385.
[] [PMID: 12824332]
Kelley, L.A.; Sternberg, M.J. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc., 2009, 4(3), 363-371.
[] [PMID: 19247286]
Laskowski, R.A. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst., 1993, 26(2), 283-291.
Jones, D.T.; Taylor, W.R.; Thornton, J.M. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci., 1992, 8(3), 275-282.
[] [PMID: 1633570]
Felsenstein, J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 1985, 39(4), 783-791.
[] [PMID: 28561359]
Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol., 2016, 33(7), 1870-1874.
[] [PMID: 27004904]
Melo, F.; Feytmans, E. Assessing protein structures with a non-local atomic interaction energy. J. Mol. Biol., 1998, 277(5), 1141-1152.
[] [PMID: 9571028]
Eisenberg, D.; Lüthy, R.; Bowie, J.U. VERIFY3D: assessment of protein models with three-dimensional profiles. Methods Enzymol., 1997, 277, 396-404.
[] [PMID: 9379925]
Velankar, S. PDBe: protein data bank in Europe. Nucleic Acids Res., 2009, 38(Database issue), D308-D317.
[] [PMID: 19858099]
The PyMOL Molecular Graphics System Version 1.8 Schrödinger, LLC, . 2015.
Marchler-Bauer, A. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res., 2016, 45(D1), D200-D203.
[] [PMID: 27899674]
Malle, D.; Iwamoto, H.; Katsuya, Y.; Utsumi, S.; Mikami, B. Crystal structure of pullulanase type I from Bacillus subtilis str. 168 in complex with maltose and alpha-cyclodextrin. (to be published),
Farber, G.K.; Petsko, G.A. The evolution of α/β barrel enzymes. Trends Biochem. Sci., 1990, 15(6), 228-234.
[] [PMID: 2200166]

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