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Biologia

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Volume 63, Issue 6

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Role of the phenylalanine 260 residue in defining product profile and alcoholytic activity of the α-amylase AmyA from Thermotoga maritima

Juanita Damián-Almazo
  • Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos, 62271, México
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/ Agustin López-Munguía
  • Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos, 62271, México
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/ Xavier Soberón-Mainero
  • Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos, 62271, México
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/ Gloria Saab-Rincón
  • Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos, 62271, México
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Published Online: 2008-12-04 | DOI: https://doi.org/10.2478/s11756-008-0170-4

Abstract

Some α-amylases besides catalyzing the hydrolysis of α-1,4 glycosidic bonds in starch are also capable of carrying out some transglycosylation activity. The importance of aromatic residues near the catalytic site in determining the ratio of these two competing activities has been remarked in the past. In the present work we investigated the role of residue 260 in the product profile of the α-amylase AmyA from Thermotoga maritima. This phenylalanine residue, two positions after the glutamic acid/base catalyst was substituted by both tryptophan and glycine residues, showing opposite behaviors. The tryptophan mutant displayed a very similar product profile pattern to that of the wild-type enzyme; while the mutant Phe260Gly showed a higher transglycosylation/hydrolysis ratio. When the Phe260Trp mutation was constructed in the context of His222Gln, a mutant we have already reported with an increased transglycosylation/hydrolysis ratio and a higher alcoholysis activity, the resultant enzyme showed an apparent higher hydrolysis/transglycosylation ratio and a change to shorter products pattern than the single mutant enzyme, still maintaining the increased alcoholytic activity provided by the His222Gln mutation. The mutant Phe260Gly, on the other hand showed by itself a higher alcoholytic activity, similar to that of the His222Gln mutant.

Keywords: alcoholysis; alkyl glycoside; α-amylase; site-directed mutagenesis; transglycosylation

  • [1] Allen J.D. & Thoma J.A. 1976. Subsite mapping of enzymes. Application of the depolymerase computer model to two α-amylases. Biochem. J. 159: 121–132. Google Scholar

  • [2] Brzozowski A.M. & Davies G.J. 1997. Structure of the Aspergillus oryzae α-amylase complexed with the inhibitor acarbose at 2.0 Å resolution. Biochemistry 36: 10837–10845. http://dx.doi.org/10.1021/bi970539iGoogle Scholar

  • [3] Brzozowski A.M., Lawson D.M., Turkenburg J.P., Bisgaard-Frantzen H., Svendsen A., Borchert T.V., Dauter Z., Wilson K.S. & Davies G.J. 2000. Structural analysis of a chimeric bacterial α-amylase. High-resolution analysis of native and ligand complexes. Biochemistry 39: 9099–9107. http://dx.doi.org/10.1021/bi0000317CrossrefGoogle Scholar

  • [4] Crabb D.W. & Mitchinson C. 1997. Enzymes involved in the processing of starch to sugars. Trends Biotechnol. 15: 349–352. http://dx.doi.org/10.1016/S0167-7799(97)01082-2CrossrefGoogle Scholar

  • [5] Damian-Almazo J.Y., Moreno A., Lopez-Munguia A., Sobreron X., Gonzalez-Munoz F. & Saab-Rincon G. 2008. Enhancement of the alcoholytic activity of α-amylase AmyA from Thermotoga maritima MSB8 (DSM 3109) by site directed mutagenesis. Appl. Environ. Microbiol. 74: 5168–5177. http://dx.doi.org/10.1128/AEM.00121-08CrossrefWeb of ScienceGoogle Scholar

  • [6] del-Rio G., Morett E. & Soberon X. 1997. Did cyclodextrin glycosyltransferases evolve from α-amylases? FEBS Lett. 416: 221–224. http://dx.doi.org/10.1016/S0014-5793(97)01192-7CrossrefGoogle Scholar

  • [7] Fogarty W.M. 1983. Microbial amylases, pp. 1–92. In: Fogarty W.M. (ed.), Microbial Enzymes and Biotechnology, Applied Science Publishers Ltd., New York. Google Scholar

  • [8] Friedberg F. 1983. On the primary structure of amylases. FEBS Lett. 152: 139–140. http://dx.doi.org/10.1016/0014-5793(83)80365-2CrossrefGoogle Scholar

  • [9] Janecek S., MacGregor E.A. & Svensson B. 1995. Characteristic differences in the primary structure allow discrimination of cyclodextrin glucanotransferases from α-amylases. Biochem. J. 305: 685–686. Google Scholar

  • [10] Janecek S., Svensson B. & Henrissat B. 1997. Domain evolution in the α-amylase family. J. Mol. Evol. 45: 322–331. http://dx.doi.org/10.1007/PL00006236CrossrefGoogle Scholar

  • [11] Jespersen H.M., MacGregor E.A., Henrissat B., Sierks M.R. & Svensson B. 1993. Starch-and glycogen-debranching and branching enzymes: prediction of structural features of the catalytic (β/α)s-barrel domain and evolutionary relationship to other amylolytic enzymes. J. Prot. Chem. 12: 791–805. http://dx.doi.org/10.1007/BF01024938CrossrefGoogle Scholar

  • [12] Jorgensen S., Vorgias C.E. & Antranikian G. 1997. Cloning, sequencing, characterization, and expression of an extracellular α-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis. J. Biol. Chem. 272: 16335–16342. http://dx.doi.org/10.1074/jbc.272.26.16335CrossrefGoogle Scholar

  • [13] Kim T.J., Kim M.J., Kim B.C., Kim J.C., Cheong T.K., Kim J.W. & Park K.H. 1999. Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain. Appl. Environ. Microbiol. 65: 1644–1651. Google Scholar

  • [14] Kim T.J., Park C.S., Cho H.Y., Cha S.S., Kim J.S., Lee S.B., Moon T.W., Kim J.W., Oh B.H. & Park K.H. 2000. Role of the glutamate 332 residue in the transglycosylation activity of Thermus maltogenic amylase. Biochemistry 39: 6773–6780. http://dx.doi.org/10.1021/bi992575iCrossrefGoogle Scholar

  • [15] Kondo H., Nakatani H., Matsuno R. & Hiromi K. 1980. Product distribution in amylase-catalyzed hydrolysis of amylose. Comparison of experimental results with theoretical predictions. J. Biochem. 87: 1053–1070. Google Scholar

  • [16] Kuriki T., Kaneko H., Yanase M., Takata H., Shimada J., Handa S., Takada T., Umeyama H. & Okada S. 1996. Controlling substrate preference and transglycosylation activity of neopullulanase by manipulating steric constraint and hydrophobicity in active center. J. Biol. Chem. 271: 17321–17329. http://dx.doi.org/10.1074/jbc.271.29.17321CrossrefGoogle Scholar

  • [17] Liebl W., Stemplinger I. & Ruile P. 1997. Properties and gene structure of the Thermotoga maritima α-amylase AmyA, a putative lipoprotein of a hyperthermophilic bacterium. J. Bacteriol. 179: 941–948. Google Scholar

  • [18] Lim W.J., Park S.R., An C.L., Lee J.Y., Hong S.Y., Shin E.C., Kim E.J., Kim J.O., Kim H. & Yun H.D. 2003. Cloning and characterization of a thermostable intracellular α-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8. Res. Microbiol. 154: 681–687. http://dx.doi.org/10.1016/j.resmic.2003.09.005CrossrefGoogle Scholar

  • [19] Matsui I., Yoneda S., Ishikawa K., Miyairi S., Fukui S., Umeyama H. & Honda K. 1994. Roles of the aromatic residues conserved in the active center of Saccharomycopsis α-amylase for transglycosylation and hydrolysis activity. Biochemistry 33: 451–458. http://dx.doi.org/10.1021/bi00168a009CrossrefGoogle Scholar

  • [20] Mosi R., He S., Uitdehaag J., Dijkstra B.W. & Withers S.G. 1997. Trapping and characterization of the reaction intermediate in cyclodextrin glycosyltransferase by use of activated substrates and a mutant enzyme. Biochemistry 36: 9927–9934. http://dx.doi.org/10.1021/bi970618uCrossrefGoogle Scholar

  • [21] Nakajima R., Imanaka T. & Aiba S. 1986. Comparison of amino acid sequences of eleven different α-amylases. Appl. Microbiol. Biotechnol. 23: 355–360. http://dx.doi.org/10.1007/BF00257032CrossrefGoogle Scholar

  • [22] Pujadas G. & Palau J. 2001. Evolution of α-amylases: architectural features and key residues in the stabilization of the (β/α)8 scaffold. Mol. Biol. Evol. 18: 38–54. Google Scholar

  • [23] Rey M.W., Brown K.M., Golightly E.J., Fuglsang C.C., Nielsen B.R., Hendriksen H.V., Butterworth A. & Xu F. 2003. Cloning, heterologous expression, and characterization of Thielavia terrestris glucoamylase. Appl. Biochem. Biotechnol. 111: 153–166. http://dx.doi.org/10.1385/ABAB:111:3:153CrossrefGoogle Scholar

  • [24] Rivera M.H., Lopez-Munguia A., Soberon X. & Saab-Rincon G. 2003. α-Amylase from Bacillus licheniformis mutants near to the catalytic site: effects on hydrolytic and transglycosylation activity. Protein Eng. 16: 505–514. http://dx.doi.org/10.1093/protein/gzg060CrossrefGoogle Scholar

  • [25] Robyt J.F. & French D. 1967. Multiple attach hypothesis of α-amylase action: action of porcine pancreatic, human salivary, and Aspergillus oryzae α-amylases. Arch. Biochem. Biophys. 122: 8–16. http://dx.doi.org/10.1016/0003-9861(67)90118-XCrossrefGoogle Scholar

  • [26] Rogers J.C. 1985. Conserved amino acid sequence domains in α-amylases from plants, mammals, and bacteria. Biochem. Biophys. Res. Comm. 128: 470–476. http://dx.doi.org/10.1016/0006-291X(85)91702-4CrossrefGoogle Scholar

  • [27] Saab-Rincon G., del-Rio G., Santamaria R.I., Lopez-Munguia A. & Soberon X. 1999. Introducing transglycosylation activity in a liquefying α-amylase. FEBS Lett. 453: 100–106. http://dx.doi.org/10.1016/S0014-5793(99)00671-7CrossrefGoogle Scholar

  • [28] Sarkar G. & Sommer S.S. 1990. The “megaprimer” method of site-directed mutagenesis. Biotechniques 8: 404–407. Google Scholar

  • [29] Suganuma T., Ohnishi M., Hiromi K. & Nagahama T. 1996. Elucidation of the subsite structure of bacterial saccharifying α-amylase and its mode of degradation of maltose. Carbohydr. Res. 282: 171–180. http://dx.doi.org/10.1016/0008-6215(95)00365-7CrossrefGoogle Scholar

  • [30] Svensson B. 1988. Regional distant sequence homology between amylases, α-glucosidases and transglucanosylases. FEBS Lett. 230: 72–76. http://dx.doi.org/10.1016/0014-5793(88)80644-6CrossrefGoogle Scholar

  • [31] Uitdehaag J.C.M., Mosi R., Kalk K.H., van der Veen B.A., Dijkhuizen L., Withers S.G. & Dijkstra B.W. 1999. X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the α-amylase family. Nat. Struct. Biol. 6: 432–436. http://dx.doi.org/10.1038/8235CrossrefGoogle Scholar

  • [32] van der Maarel M.J., van der Veen B., Uitdehaag J.C., Leemhuis H. & Dijkhuizen L. 2002. Properties and applications of starch-converting enzymes of the α-amylase family. J. Biotechnol. 94: 137–155. http://dx.doi.org/10.1016/S0168-1656(01)00407-2CrossrefGoogle Scholar

  • [33] van der Veen B.A., Leemhuis H., Kralj S., Uitdehaag J.C.M., Dijkstra B.W. & Dijkhuizen L. 2001. Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextringlycosyltransferase. J. Biol. Chem. 276: 44557–44562. http://dx.doi.org/10.1074/jbc.M107533200CrossrefGoogle Scholar

  • [34] Vihinen M. & Mantsala P. 1989. Microbial amylolytic enzymes. Crit. Rev. Biochem. Mol. Biol. 24: 329–418. http://dx.doi.org/10.3109/10409238909082556CrossrefGoogle Scholar

About the article

Published Online: 2008-12-04

Published in Print: 2008-12-01


Citation Information: Biologia, Volume 63, Issue 6, Pages 1035–1043, ISSN (Online) 1336-9563, ISSN (Print) 0006-3088, DOI: https://doi.org/10.2478/s11756-008-0170-4.

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© 2008 Slovak Academy of Sciences. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

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