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Protein & Peptide Letters

Editor-in-Chief

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

Research Article

Denaturant Induced Equilibrium Unfolding and Conformational Transitional Studies of Germinated Fenugreek β-Amylase Revealed Molten Globule like State at Low pH

Author(s): Dinesh Chand Agrawal, Anjali Yadav, Mohd. Asim Khan, Suman Kundu* and Arvind M. Kayastha*

Volume 27, Issue 10, 2020

Page: [1046 - 1057] Pages: 12

DOI: 10.2174/0929866527666200403082721

Price: $65

Abstract

Background: β-Amylase (EC 3.2.1.2) is a maltogenic enzyme, which releases β-maltose from the non-reducing end of the substrates. The enzyme plays important roles for the production of vaccine, maltiol and maltose rich syrups. Apart from these applications the enzyme protects cells from abiotic as well as oxidative damage. The enzyme is βwell characterized in βplants and microbes and crystal structures of β-amylases βhave been βobtained from sweet potato, soybean and Bacillus cereus.

Objective: Find out correlation between structural and functional stability induced by change in pH, temperature and chaotropes.

Methods: Activity, intrinsic fluorescence, extrinsic fluorescence, near- and far- ultraviolet circular dichroism spectroscopic measurements were performed.

Results: Peaks about 208 nm and 222 nm obtained by near-ultraviolet circular dichroism correspond to α-helix whereas peak at 215 nm shows presence of β-sheet. At pH 2.0, absence of tertiary structures, exposed of hydrophobic regions and presence of substantial secondary structures, revealed the existence of molten globule like state. Temperature induced denaturation studies showed that the enzyme was stable up to 75 ºC and the process was found to be irreversible in nature. Chaotropes dependent equilibrium unfolding studies revealed that at low concentration of chaotropes, ellipticity and intrinsic fluorescence βintensity were βdecreased βwhereas βenzymatic activity remained unchanged, which revealed fenugreek β-amylase is multi-domains enzyme and catalytic βdomain βis more βstable compare to non-catalytic domain. Moreover, the transition was sigmoidal and non-coincidental.

Conclusion: Results indicate the probable existence of intermediate states that might perform significant role in physiological process and biotechnological applications.

Keywords: β-Amylase, cooperative, intermediate, molten globule, fenugreek, circular dichroism.

Graphical Abstract
[1]
Goesaert, H.; Slade, L.; Levine, H.; Delcour, J.A. Amylases and bread firming an integrated view. J. Cereal Sci., 2009, 50(3), 345-352.
[http://dx.doi.org/10.1016/j.jcs.2009.04.010]
[2]
Hyun, H.H.; Zeikus, J.G. General biochemical characterization of thermostable extracellular β-amylase from Clostridium thermosulfurogenes. Appl. Environ. Microbiol., 1985, 49(5), 1162-1167.
[http://dx.doi.org/10.1128/AEM.49.5.1162-1167.1985 ] [PMID: 16346789]
[3]
Nehete, P.N.; Shah, N.K.; Ramamurthy, V.; Kothari, R.M. An optimized protocol for the production of high purity maltose. World J. Microbiol. Biotechnol., 1992, 8(4), 446-450.
[http://dx.doi.org/10.1007/BF01198763 ] [PMID: 24425521]
[4]
Ziegler, P. Cereal β-amylases. J. Cereal Sci., 1999, 29(3), 195-204.
[http://dx.doi.org/10.1006/jcrs.1998.0238]
[5]
Henkel, J. Sugar substitutes. Americans opt for sweetness and lite. FDA Consum., 1999, 33(6), 12-16.
[PMID: 10628311]
[6]
Das, R.; Kayastha, A.M. An antioxidant rich novel β-amylase from peanuts (Arachis hypogaea): Its purification, biochemical characterization and potential applications. Int. J. Biol. Macromol., 2018, 111, 148-157.
[http://dx.doi.org/10.1016/j.ijbiomac.2017.12.130 ] [PMID: 29305882]
[7]
Chandrika, C.; Vijayashree, C.; Granthali, S.; Rajath, S.; Nagananda, G.S. A comparative kinetic study on β-amylase and its antioxidant property in germinated and non-germinated seeds of Glycine max. L. Biotechnol Res., 2013, 4(4), 13-24.
[8]
Nandi, S. Das, G.; Sen-Mandi, S. β-Amylase activity as an index for germination potential in rice. Ann. Bot., 1995, 75(5), 463-467.
[http://dx.doi.org/10.1006/anbo.1995.1046]
[9]
Bilderback, D.E. Amylases from aleurone layers and starchy endosperm of barley seeds. Plant Physiol., 1974, 53(3), 480-484.
[http://dx.doi.org/10.1104/pp.53.3.480 ] [PMID: 16658728]
[10]
Cheong, C.G.; Eom, S.H.; Chang, C.; Shin, D.H.; Song, H.K.; Min, K.; Moon, J.H.; Kim, K.K.; Hwang, K.Y.; Suh, S.W. Crystallization, molecular replacement solution, and refinement of tetrameric β-amylase from sweet potato. Proteins, 1995, 21(2), 105-117.
[http://dx.doi.org/10.1002/prot.340210204 ] [PMID: 7777485]
[11]
Hara, M.; Sawada, T.; Ito, A.; Ito, F.; Kuboi, T. A major β-amylase expressed in radish taproots. Food Chem., 2009, 114(2), 523-528.
[http://dx.doi.org/10.1016/j.foodchem.2008.09.082]
[12]
Mikami, B.; Degano, M.; Hehre, E.J.; Sacchettini, J.C. Crystal structures of soybean β-amylase reacted with β-maltose and maltal: Active site components and their apparent roles in catalysis. Biochemistry, 1994, 33(25), 7779-7787.
[http://dx.doi.org/10.1021/bi00191a005 ] [PMID: 8011643]
[13]
Doehlert, D.C. Duke, S.H.; Anderson, L. β-amylases from alfalfa (Medicago sativa L.) roots. Plant Physiol., 1982, 69(5), 1096-1102.
[http://dx.doi.org/10.1104/pp.69.5.1096 ] [PMID: 16662350]
[14]
Ray, R.R.; Jana, S.C.; Nanda, G. Biochemical approaches of increasing thermostability of β-amylase from Bacillus megaterium B6. FEBS Lett., 1994, 356(1), 30-32.
[http://dx.doi.org/10.1016/0014-5793(94)01227-X ] [PMID: 7988714]
[15]
Adachi, M.; Mikami, B.; Katsube, T.; Utsumi, S. Crystal structure of recombinant soybean β-amylase complexed with β-cyclodextrin. J. Biol. Chem., 1998, 273(31), 19859-19865.
[http://dx.doi.org/10.1074/jbc.273.31.19859 ] [PMID: 9677422]
[16]
Mikami, B.; Adachi, M.; Kage, T.; Sarikaya, E.; Nanmori, T.; Shinke, R.; Utsumi, S. Structure of raw starch-digesting Bacillus cereus β-amylase complexed with maltose. Biochemistry, 1999, 38(22), 7050-7061.
[http://dx.doi.org/10.1021/bi9829377 ] [PMID: 10353816]
[17]
Balcão, V.M.; Vila, M.M. Structural and functional stabilization of protein entities: State-of-the-art. Adv. Drug Deliv. Rev., 2015, 93, 25-41.
[http://dx.doi.org/10.1016/j.addr.2014.10.005 ] [PMID: 25312675]
[18]
Singh, N.; Kumar, R.; Jagannadham, M.V.; Kayastha, A.M. Evidence for a molten globule state in Cicer α-galactosidase induced by pH, temperature, and guanidine hydrochloride. Appl. Biochem. Biotechnol., 2013, 169(8), 2315-2325.
[http://dx.doi.org/10.1007/s12010-013-0163-9 ] [PMID: 23446984]
[19]
Onuchic, J.N.; Socci, N.D.; Luthey-Schulten, Z.; Wolynes, P.G. Protein folding funnels: The nature of the transition state ensemble. Fold. Des., 1996, 1(6), 441-450.
[http://dx.doi.org/10.1016/S1359-0278(96)00060-0 ] [PMID: 9080190]
[20]
Dill, K.A.; Chan, H.S. From Levinthal to pathways to funnels. Nat. Struct. Biol., 1997, 4(1), 10-19.
[http://dx.doi.org/10.1038/nsb0197-10 ] [PMID: 8989315]
[21]
Semisotnov, G.V.; Rodionova, N.A.; Razgulyaev, O.I.; Uversky, V.N.; Gripas’, A.F.; Gilmanshin, R.I. Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers, 1991, 31(1), 119-128.
[http://dx.doi.org/10.1002/bip.360310111 ] [PMID: 2025683]
[22]
van der Goot, F.G.; González-Mañas, J.M.; Lakey, J.H.; Pattus, F.A. ‘molten-globule’ membrane-insertion intermediate of the pore-forming domain of colicin A. Nature, 1991, 354(6352), 408-410.
[http://dx.doi.org/10.1038/354408a0 ] [PMID: 1956406]
[23]
Ren, J.; Kachel, K.; Kim, H.; Malenbaum, S.E.; Collier, R.J.; London, E. Interaction of diphtheria toxin T domain with molten globule-like proteins and its implications for translocation. Science, 1999, 284(5416), 955-957.
[http://dx.doi.org/10.1126/science.284.5416.955 ] [PMID: 10320374]
[24]
Martin, J.; Langer, T.; Boteva, R.; Schramel, A.; Horwich, A.L.; Hartl, F-U. Chaperonin-mediated protein folding at the surface of groEL through a ‘molten globule’-like intermediate. Nature, 1991, 352(6330), 36-42.
[http://dx.doi.org/10.1038/352036a0 ] [PMID: 1676490]
[25]
Agrawal, D.C.; Dwevedi, A.; Kayastha, A.M. Biochemical and thermodynamic characterization of de novo synthesized β-amylase from fenugreek. Int. J. Biol. Macromol., 2019, 130, 786-797.
[http://dx.doi.org/10.1016/j.ijbiomac.2019.02.162 ] [PMID: 30831171]
[26]
Srivastava, G. Kayastha, A.M. α-amylase from starchless seeds of Trigonella foenum-graecum and its localization in germinating seeds. PLoS One, 2014, 9(2), e88697.
[http://dx.doi.org/10.1371/journal.pone.0088697 ] [PMID: 24551136]
[27]
Bernfeld, P. Amylases, α and β. Methods Enzymol., 1955, 1, 149-158.
[http://dx.doi.org/10.1016/0076-6879(55)01021-5]
[28]
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72(2), 248-254.
[http://dx.doi.org/10.1016/0003-2697(76)90527-3 ] [PMID: 942051]
[29]
Uehara, K.; Mannen, S. Interaction of sweet potato β-amylase with its reaction product, maltose. J. Biochem., 1979, 85(1), 105-113.
[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a132299 ] [PMID: 153906]
[30]
Greenfield, N.J. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc., 2006, 1(6), 2876-2890.
[http://dx.doi.org/10.1038/nprot.2006.202 ] [PMID: 17406547]
[31]
Singh, K.; Shandilya, M.; Kundu, S.; Kayastha, A.M. Heat, acid and chemically induced unfolding pathways, conformational stability and structure-function relationship in wheat α-amylase. PLoS One, 2015, 10(6), e0129203.
[http://dx.doi.org/10.1371/journal.pone.0129203 ] [PMID: 26053142]
[32]
Dubey, V.K.; Jagannadham, M.V. Differences in the unfolding of procerain induced by pH, guanidine hydrochloride, urea, and temperature. Biochemistry, 2003, 42(42), 12287-12297.
[http://dx.doi.org/10.1021/bi035047m ] [PMID: 14567690]
[33]
Dwevedi, A.; Dubey, V.K.; Jagannadham, M.V.; Kayastha, A.M. Insights into pH-induced conformational transition of β-galactosidase from Pisum sativum leading to its multimerization. Appl. Biochem. Biotechnol., 2010, 162(8), 2294-2312.
[http://dx.doi.org/10.1007/s12010-010-9003-3 ] [PMID: 20549573]
[34]
Kelly, S.M.; Price, N.C. The use of circular dichroism in the investigation of protein structure and function. Curr. Protein Pept. Sci., 2000, 1(4), 349-384.
[http://dx.doi.org/10.2174/1389203003381315 ] [PMID: 12369905]
[35]
Muzammil, S.; Kumar, Y.; Tayyab, S. Molten globule-like state of human serum albumin at low pH. Eur. J. Biochem., 1999, 266(1), 26-32.
[http://dx.doi.org/10.1046/j.1432-1327.1999.00810.x ] [PMID: 10542047]
[36]
Golczak, M.; Kicinska, A.; Bandorowicz-Pikula, J.; Buchet, R.; Szewczyk, A.; Pikula, S. Acidic pH-induced folding of annexin VI is a prerequisite for its insertion into lipid bilayers and formation of ion channels by the protein molecules. FASEB J., 2001, 15(6), 1083-1085.
[PMID: 11292675]
[37]
Carneiro, F.A.; Ferradosa, A.S.; Da Poian, A.T. Low pH-induced conformational changes in vesicular stomatitis virus glycoprotein involve dramatic structure reorganization. J. Biol. Chem., 2001, 276(1), 62-67.
[http://dx.doi.org/10.1074/jbc.M008753200 ] [PMID: 11024041]
[38]
Clackson, T.; Wells, J.A. A hot spot of binding energy in a hormone-receptor interface. Science, 1995, 267(5196), 383-386.
[http://dx.doi.org/10.1126/science.7529940 ] [PMID: 7529940]
[39]
Bogan, A.A.; Thorn, K.S. Anatomy of hot spots in protein interfaces. J. Mol. Biol., 1998, 280(1), 1-9.
[http://dx.doi.org/10.1006/jmbi.1998.1843 ] [PMID: 9653027]
[40]
Tsai, C.J.; Lin, S.L.; Wolfson, H.J.; Nussinov, R. Studies of protein-protein interfaces: A statistical analysis of the hydrophobic effect. Protein Sci., 1997, 6(1), 53-64.
[http://dx.doi.org/10.1002/pro.5560060106 ] [PMID: 9007976]
[41]
Park, S.J.; Borin, B.N.; Martinez-Yamout, M.A.; Dyson, H.J. The client protein p53 adopts a molten globule-like state in the presence of Hsp90. Nat. Struct. Mol. Biol., 2011, 18(5), 537-541.
[http://dx.doi.org/10.1038/nsmb.2045 ] [PMID: 21460846]
[42]
Fitter, J.; Haber-Pohlmeier, S. Structural stability and unfolding properties of thermostable bacterial α-amylases: A comparative study of homologous enzymes. Biochemistry, 2004, 43(30), 9589-9599.
[http://dx.doi.org/10.1021/bi0493362 ] [PMID: 15274613]
[43]
Kishore, D.; Kundu, S.; Kayastha, A.M. Thermal, chemical and pH induced denaturation of a multimeric β-galactosidase reveals multiple unfolding pathways. PLoS One, 2012, 7(11), e50380.
[http://dx.doi.org/10.1371/journal.pone.0050380 ] [PMID: 23185611]
[44]
Duy, C.; Fitter, J. How aggregation and conformational scrambling of unfolded states govern fluorescence emission spectra. Biophys. J., 2006, 90(10), 3704-3711.
[http://dx.doi.org/10.1529/biophysj.105.078980 ] [PMID: 16500981]
[45]
Sabate, R.; Rodriguez-Santiago, L.; Sodupe, M.; Saupe, S.J.; Ventura, S. Thioflavin-T excimer formation upon interaction with amyloid fibers. Chem. Commun. (Camb.), 2013, 49(51), 5745-5747.
[http://dx.doi.org/10.1039/c3cc42040j ] [PMID: 23687656]
[46]
Hortschansky, P.; Schroeckh, V.; Christopeit, T.; Zandomeneghi, G.; Fändrich, M. The aggregation kinetics of Alzheimer’s β-amyloid peptide is controlled by stochastic nucleation. Protein Sci., 2005, 14(7), 1753-1759.
[http://dx.doi.org/10.1110/ps.041266605 ] [PMID: 15937275]
[47]
Duy, C.; Fitter, J. Thermostability of irreversible unfolding α-amylases analyzed by unfolding kinetics. J. Biol. Chem., 2005, 280(45), 37360-37365.
[http://dx.doi.org/10.1074/jbc.M507530200 ] [PMID: 16150692]
[48]
Kaur, P.K.; Supin, J.S.; Rashmi, S.; Singh, S. Chemical- and thermal-induced unfolding of Leishmania donovani ribose-5-phosphate isomerase B: A single-tryptophan protein. Appl. Biochem. Biotechnol., 2014, 173(7), 1870-1884.
[http://dx.doi.org/10.1007/s12010-014-0973-4 ] [PMID: 24907042]
[49]
Canchi, D.R.; Paschek, D.; García, A.E. Equilibrium study of protein denaturation by urea. J. Am. Chem. Soc., 2010, 132(7), 2338-2344.
[http://dx.doi.org/10.1021/ja909348c ] [PMID: 20121105]

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