SpringerLink
Log in

Importance of Trp139 in the product specificity of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04 (Bst-MFA) randomly cleaves the α-1,4 glycosidic linkages of starch to produce predominantly maltopentaose and maltohexaose. The three-dimensional co-crystal structure of Bst-MFA with acarbose highlighted the stacking interactions between Trp139 and the substrate in subsites − 5 and − 6. Interactions like this are thought to play a critical role in maltopentaose/maltohexaose production. A site-directed mutagenesis approach was used to test this hypothesis. Replacement of Trp139 by alanine, leucine, or tyrosine dramatically increased maltopentaose production and reduced maltohexaose production. Oligosaccharide degradation indicated that these mutants also enhance productive binding of the substrate aglycone, leading to a high maltopentaose yield. Therefore, the aromatic stacking between Trp139 and substrate is suggested to control product specificity and the oligosaccharide cleavage pattern.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Germany)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Aghajari N, Roth M, Haser R (2002) Crystallographic evidence of a transglycosylation reaction: ternary complexes of a psychrophilic α-amylase. Biochemistry 41(13):4273–4280

    CAS  PubMed  Google Scholar 

  • Agirre J, Moroz O, Meier S, Brask J, Munch A, Hoff T, Andersen C, Wilson KS, Davies GJ (2019) The structure of the AliC GH13 α-amylase from Alicyclobacillus sp. reveals the accommodation of starch branching points in the α-amylase family. Acta Crystallogr D Struct Biol 75(Pt 1):1–7

    CAS  PubMed  PubMed Central  Google Scholar 

  • Alikhajeh J, Khajeh K, Ranjbar B, Naderi-Manesh H, Lin YH, Liu E, Guan HH, Hsieh YC, Chuankhayan P, Huang YC, Jeyaraman J, Liu MY, Chen CJ (2010) Structure of Bacillus amyloliquefaciens α-amylase at high resolution: implications for thermal stability. Acta Crystallogr Sect F Struct Biol Cryst Commun 66(Pt 2):121–129

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bai Y, Gangoiti J, Dijkstra BW, Dijkhuizen L, Pijning T (2017) Crystal structure of 4,6-α-glucanotransferase supports diet-driven evolution of GH70 enzymes from α-amylases in oral Bacteria. Structure 25(2):231–242

    CAS  PubMed  Google Scholar 

  • Bak-Jensen KS, Andre G, Gottschalk TE, Paes G, Tran V, Svensson B (2004) Tyrosine 105 and threonine 212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of barley α-amylase 1. J Biol Chem 279(11):10093–10102

    CAS  PubMed  Google Scholar 

  • Bozonnet S, Jensen MT, Nielsen MM, Aghajari N, Jensen MH, Kramhoft B, Willemoes M, Tranier S, Haser R, Svensson B (2007) The 'pair of sugar tongs' site on the non-catalytic domain C of barley α-amylase participates in substrate binding and activity. FEBS J 274(19):5055–5067

    CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1-2):248–254

    CAS  Google Scholar 

  • Brzozowski AM, Davies GJ (1997) Structure of the Aspergillus oryzae α-amylase complexed with the inhibitor acarbose at 2.0 Å resolution. Biochemistry 36(36):10837–10845

    CAS  PubMed  Google Scholar 

  • Brzozowski AM, Lawson DM, Turkenburg JP, Bisgaard-Frantzen H, Svendsen A, Borchert TV, Dauter Z, Wilson KS, Davies GJ (2000) Structural analysis of a chimeric bacterial α-amylase: high-resolution analysis of native and ligand complexes. Biochemistry 39(31):9099–9107

    CAS  PubMed  Google Scholar 

  • Chai KP, Othman NF, Teh AH, Ho KL, Chan KG, Shamsir MS, Goh KM, Ng CL (2016) Crystal structure of Anoxybacillus α-amylase provides insights into maltose binding of a new glycosyl hydrolase subclass. Sci Rep 6:23126

    CAS  PubMed  PubMed Central  Google Scholar 

  • Davies GJ, Brzozowski AM, Dauter Z, Rasmussen MD, Borchert TV, Wilson KS (2005) Structure of a Bacillus halmapalus family 13 α-amylase, BHA, in complex with an acarbose-derived nonasaccharide at 2.1 Å resolution. Acta Crystallogr D Biol Crystallogr 61(Pt 2):190–193

    PubMed  Google Scholar 

  • Dumbrepatil AB, Choi JH, Park JT, Kim MJ, Kim TJ, Woo EJ, Park KH (2010) Structural features of the Nostoc punctiforme debranching enzyme reveal the basis of its mechanism and substrate specificity. Proteins 78(2):348–356

    CAS  PubMed  Google Scholar 

  • Fujimoto Z, Takase K, Doui N, Momma M, Matsumoto T, Mizuno H (1998) Crystal structure of a catalytic-site mutant α-amylase from Bacillus subtilis complexed with maltopentaose. J Mol Biol 277(2):393–407

    CAS  PubMed  Google Scholar 

  • Gourlay LJ, Santi I, Pezzicoli A, Grandi G, Soriani M, Bolognesi M (2009) Group B streptococcus pullulanase crystal structures in the context of a novel strategy for vaccine development. J Bacteriol 191(11):3544–3552

    CAS  PubMed  PubMed Central  Google Scholar 

  • Janeček S (2002) How many conserved sequence regions are there in the α-amylase family? Biologia 11(supplement 11):29–41

    Google Scholar 

  • Janeček S, Gabriško M (2016) Remarkable evolutionary relatedness among the enzymes and proteins from the α-amylase family. Cell Mol Life Sci 73(14):2707–2725

    PubMed  Google Scholar 

  • Janeček S, Svensson B, MacGregor EA (2014) α-Amylase: an enzyme specificity found in various families of glycoside hydrolases. Cell Mol Life Sci 71(7):1149–1170

    PubMed  Google Scholar 

  • Kadziola A, Søgaard M, Svensson B, Haser R (1998) Molecular structure of a barley α-amylase-inhibitor complex: implications for starch binding and catalysis. J Mol Biol 278(1):205–217

    CAS  PubMed  Google Scholar 

  • Kanai R, Haga K, Akiba T, Yamane K, Harata K (2004) Biochemical and crystallographic analyses of maltohexaose-producing amylase from alkalophilic Bacillus sp. 707. Biochemistry 43(44):14047–14056

    CAS  PubMed  Google Scholar 

  • Kanai R, Haga K, Akiba T, Yamane K, Harata K (2006) Role of Trp140 at subsite -6 on the maltohexaose production of maltohexaose-producing amylase from alkalophilic Bacillus sp.707. Protein Sci 15(3):468–477

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kandra L, Hachem MA, Gyemant G, Kramhoft B, Svensson B (2006) Map** of barley α-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft. FEBS Lett 580(21):5049–5053

    CAS  PubMed  Google Scholar 

  • Kim TU, Gu BG, Jeong JY, Byun SM (1995) Purification and characterization of a maltotetraose-forming alkaline α-amylase from an alkalophilic Bacillus strain, GM8901. Appl Environ Microbiol 61(8):3105–3112

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kramhøft B, Bak-Jensen KS, Mori H, Juge N, Nøhr J, Svensson B (2005) Involvement of individual subsites and secondary substrate binding sites in multiple attack on amylose by Barley α-amylase. Biochemistry 44(6):1824–1832

    PubMed  Google Scholar 

  • Linden A, Mayans O, Meyer-Klaucke W, Antranikian G, Wilmanns M (2003) Differential regulation of a hyperthermophilic α-amylase with a novel (Ca, Zn) two-metal center by zinc. J Biol Chem 278(11):9875–9884

    CAS  PubMed  Google Scholar 

  • Machius M, Declerck N, Huber R, Wiegand G (1998) Activation of Bacillus licheniformis α-amylase through a disorder→order transition of the substrate-binding site mediated by a calcium–sodium–calcium metal triad. Structure 6(3):281–292

    CAS  PubMed  Google Scholar 

  • Machius M, Vértesy L, Huber R, Wiegand G (1996) Carbohydrate and protein-based inhibitors of porcine pancreatic α-amylase: structure analysis and comparison of their binding characteristics. J Mol Biol 260(3):409–421

    CAS  PubMed  Google Scholar 

  • Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Biochem 31(3):426–428

    CAS  Google Scholar 

  • Momma M (2000) Cloning and sequencing of the maltohexaose-producing amylase gene of Klebsiella pneumoniae. Biosci Biotechnol Biochem 64(2):428–431

    CAS  PubMed  Google Scholar 

  • Nakajima R, Imanaka T, Aiba S (1986) Comparison of amino acid sequences of eleven different α-amylases. Appl Microbiol Biotechnol 23(5):355–360

    CAS  Google Scholar 

  • Nielsen MM, Seo ES, Bozonnet S, Aghajari N, Robert X, Haser R, Svensson B (2008) Multi-site substrate binding and interplay in barley α-amylase 1. FEBS Lett 582(17):2567–2571

    CAS  PubMed  Google Scholar 

  • Nonaka T, Fujihashi M, Kita A, Hagihara H, Ozaki K, Ito S, Miki K (2003) Crystal structure of calcium-free α-amylase from Bacillus sp. strain KSM-K38 (AmyK38) and its sodium ion binding sites. J Biol Chem 278(27):24818–24824

    CAS  PubMed  Google Scholar 

  • Ochiai A, Sugai H, Harada K, Tanaka S, Ishiyama Y, Ito K, Tanaka T, Uchiumi T, Taniguchi M, Mitsui T (2014) Crystal structure of α-amylase from Oryza sativa: molecular insights into enzyme activity and thermostability. Biosci Biotechnol Biochem 78(6):989–997

    CAS  PubMed  Google Scholar 

  • Pan S, Ding N, Ren J, Gu Z, Li C, Hong Y, Cheng L, Holler TP, Li Z (2017) Maltooligosaccharide-forming amylase: characteristics, preparation, and application. Biotechnol Adv 35(5):619–632

    CAS  PubMed  Google Scholar 

  • Pan S, Gu Z, Ding N, Zhang Z, Chen D, Li C, Hong Y, Cheng L, Li Z (2019) Calcium and sodium ions synergistically enhance the thermostability of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04. Food Chem 283:170–176

    CAS  PubMed  Google Scholar 

  • Park JT, Song HN, Jung TY, Lee MH, Park SG, Woo EJ, Park KH (2013) A novel domain arrangement in a monomeric cyclodextrin-hydrolyzing enzyme from the hyperthermophile Pyrococcus furiosus. Biochim Biophys Acta 1834(1):380–386

    CAS  PubMed  Google Scholar 

  • Przylas I, Terada Y, Fujii K, Takaha T, Saenger W, Strater N (2000) X-ray structure of acarbose bound to amylomaltase from Thermus aquaticus: implications for the synthesis of large cyclic glucans. Eur J Biochem 267(23):6903–6913

    CAS  PubMed  Google Scholar 

  • Qian M, Haser R, Buisson G, Duée E, Payan F (1994) The active center of a mammalian α-amylase. Structure of the complex of a pancreatic α-amylase with a carbohydrate inhibitor refined to 2.2-Å resolution. Biochemistry 33(20):6284–6294

    CAS  PubMed  Google Scholar 

  • Quiocho FA, Spurlino JC, Rodseth LE (1997) Extensive features of tight oligosaccharide binding revealed in high-resolution structures of the maltodextrin transport/chemosensory receptor. Structure 5(5):997–1015

    CAS  PubMed  Google Scholar 

  • Robert X, Haser R, Gottschalk TE, Ratajczak F, Driguez H, Svensson B, Aghajari N (2003) The structure of barley α-amylase Isozyme 1 reveals a novel role of domain C in substrate recognition and binding. Structure 11(8):973–984

    CAS  PubMed  Google Scholar 

  • Robert X, Haser R, Mori H, Svensson B, Aghajari N (2005) Oligosaccharide binding to barley α-amylase 1. J Biol Chem 280(38):32968–32978

    CAS  PubMed  Google Scholar 

  • Shirai T, Igarashi K, Ozawa T, Hagihara H, Kobayashi T, Ozaki K, Ito S (2007) Ancestral sequence evolutionary trace and crystal structure analyses of alkaline α-amylase from Bacillus sp. KSM-1378 to clarify the alkaline adaptation process of proteins. Proteins 66(3):600–610

    CAS  PubMed  Google Scholar 

  • Uitdehaag JCM, Kalk KH, van der Veen BA, Dijkhuizen L, Dijkstra BW (1999) The cyclization mechanism of cyclodextrin glycosyltransferase (CGTase) as revealed by a γ-cyclodextrin-CGTase complex at 1.8 Å resolution. J Biol Chem 274(49):34868–34876

    CAS  PubMed  Google Scholar 

  • Uitdehaag JCM, Kalk KH, van der Veen BA, Dijkhuizen L, Dijkstra BW (2000) Structures of maltohexaose and maltoheptaose bound at the donor sites of cyclodextrin glycosyltransferase give insight into the mechanisms of transglycosylation activity and cyclodextrin size specificity. Biochemistry 39(26):7772–7780

    CAS  PubMed  Google Scholar 

  • van der Veen BA, Leemhuis H, Kralj S, Uitdehaag JC, Dijkstra BW, Dijkhuizen L (2001) Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin-glycosyltransferase. J Biol Chem 276(48):44557–44562

    PubMed  Google Scholar 

  • **e X, Li Y, Ban X, Zhang Z, Gu Z, Li C, Hong Y, Cheng L, ** T, Li Z (2019) Crystal structure of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04. Int J Biol Macromol 138:394–402

    CAS  PubMed  Google Scholar 

  • Yang CH, Liu WH (2004) Purification and properties of a maltotriose-producing α-amylase from Thermobifida fusca. Enzym Microb Technol 35(2-3):254–260

    CAS  Google Scholar 

  • Yoshioka Y, Hasegawa K, Matsuura Y, Katsube Y, Kubota M (1997) Crystal structures of a mutant maltotetraose-forming exo-amylase cocrystallized with maltopentaose. J Mol Biol 271(4):619–628

    CAS  PubMed  Google Scholar 

Download references

Funding

This work was financially supported by the National Natural Science Foundation of China (No. 31722040, 31571882), China Postdoctoral Science Foundation (No. 2018M632233), the Natural Science Foundation of Jiangsu Province (BK20180606), and the Jiangsu province “Collaborative Innovation Center of Food Safety and Quality Control” industry development program.

Author information

Authors and Affiliations

  1. School of Food Science and Technology, Jiangnan University, Wuxi, 214122, People’s Republic of China

    **aofang **e, Gaoyuan Qiu, Ziqian Zhang, **aofeng Ban, Zhengbiao Gu, Caiming Li, Yan Hong, Li Cheng & Zhaofeng Li

  2. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Zhengbiao Gu, Caiming Li, Yan Hong, Li Cheng & Zhaofeng Li

  3. Collaborative Innovation Center of Food Safety and Quality Control, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Zhengbiao Gu, Caiming Li, Yan Hong, Li Cheng & Zhaofeng Li

Authors
  1. **aofang **e
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gaoyuan Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ziqian Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. **aofeng Ban
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhengbiao Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Caiming Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yan Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Li Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zhaofeng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zhaofeng Li.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 319 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

**e, X., Qiu, G., Zhang, Z. et al. Importance of Trp139 in the product specificity of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04. Appl Microbiol Biotechnol 103, 9433–9442 (2019). https://doi.org/10.1007/s00253-019-10194-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-019-10194-6

Keywords

Search

Navigation