Skip to main content
SpringerLink
Log in

Modulating the function of a β-1,3-glucanosyltransferase to that of an endo-β-1,3-glucanase by structure-based protein engineering

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

A glycoside hydrolase (GH) family 17 β-1,3-glucanosyltransferase (RmBgt17A) from Rhizomucor miehei CAU432 (CGMCC No. 4967) shared very low sequence homology (∼20 % identity) with that of other β-1,3-glucanases, despite their similar structural folds. Structural comparison and sequence alignment between RmBgt17A and GH family 17 β-1,3-glucanases suggested important roles for three residues (Tyr102, Trp157, and Glu158) located in the substrate-binding cleft of RmBgt17A in transglycosylation activity. A series of site-directed mutagenesis studies indicated that a single Glu-to-Ala mutation (E158A) modulates the function of RmBgt17A to that of a β-1,3-glucanase. Mutant E158A exhibited high hydrolytic activity (39.95 U/mg) toward reduced laminarin, 348.5-fold higher than the wild type. Optimal pH and temperature of the purified RmBgt17A-E158A were 4.5 and 55 °C, respectively. TLC analysis suggested that RmBgt17A-E158A is an endo-β-1,3-glucanase. Our study provides novel insight into protein engineering of the substrate-binding cleft of glycoside hydrolases to modulate the function of transglycosylation and hydrolysis.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

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

  • Akiyama T, Shibuya N, Hrmova M, Fincher GB (1997) Purification and characterization of a (1 → 3)-β-D-glucan endohydrolase from rice (Oryza sativa) bran. Carbohydr Res 297:365–374

    Article  CAS  PubMed  Google Scholar 

  • Arab-Jaziri F, Bissaro B, Dion M, Saurel O, Harrison D, Ferreira F, Milon A, Tellier C, Faure R, O’Donohue MJ (2013) Engineering transglycosidase activity into a GH51 α-l-arabinofuranosidase. New Biotechnol 30:536–544

    Article  CAS  Google Scholar 

  • Bezsonov EE, Groenning M, Galzitskaya OV, Gorkovskii AA, Semisotnov GV, Selyakh IO, Ziganshin RH, Rekstina VV, Kudryashova IB, Kuznetsov SA, Kulaev IS, Kalebina TS (2013) Amyloidogenic peptides of yeast cell wall glucantransferase Bgl2p as a model for the investigation of its pH-dependent fibril formation. Prion 7:175–184

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Blättel V, Larisika M, Pfeiffer P, Nowak C, Eich A, Eckelt J, Konig H (2011) β-1,3-glucanase from Delftia tsuruhatensis strain MV01 and its potential application in vinification. Appl Environ Microbiol 77:983–990

    Article  PubMed Central  PubMed  Google Scholar 

  • Bond CS, Schuttelkopf AW (2009) ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallogr D Biol Crystallogr 65:510–512

    Article  CAS  PubMed  Google Scholar 

  • Brooks BR, Brooks CL, Mackerell AD, Nilsson L, Petrella RJ, Roux B, Won Y, Archontis G, Bartels C, Boresch S, Caflisch A, Caves L, Cui Q, Dinner AR, Feig M, Fischer S, Gao J, Hodoscek M, Im W, Kuczera K, Lazaridis T, Ma J, Ovchinnikov V, Paci E, Pastor RW, Post CB, Pu JZ, Schaefer M, Tidor B, Venable RM, Woodcock HL, Wu X, Yang W, York DM, Karplus M (2009) CHARMM: the biomolecular simulation program. J Comput Chem 30:1545–1614

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chen L, Garrett TP, Fincher GB, Hoj PB (1995) A tetrad of ionizable amino acids is important for catalysis in barley β-glucanases. J Biol Chem 270:8093–8101

    Article  CAS  PubMed  Google Scholar 

  • Cheng R, Chen J, Yu X, Wang Y, Wang S, Zhang J (2013) Recombinant production and characterization of full-length and truncated β-1,3-glucanase PglA from Paenibacillus sp. S09. BMC Biotechnol 13:105

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Churngchow N, Suntaro A, Wititsuwannnakul R (1995) β-1,3-glucanase isozymes from the latex of Hevea brasiliensis. Phytochemistry 39:505–509

    Article  CAS  Google Scholar 

  • Cota J, Alvarez TM, Citadini AP, Santos CR, de Oliveira NM, Oliveira RR, Pastore GM, Ruller R, Prade RA, Murakami MT, Squina FM (2011) Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-β-1,3-glucanase from Thermotoga petrophila. Biochem Biophys Res Commun 406:590–594

    Article  CAS  PubMed  Google Scholar 

  • Daggett V, Fersht AR (2003) Is there a unifying mechanism for protein folding? Trends Biochem Sci 28:18–25

    Article  CAS  PubMed  Google Scholar 

  • Davies GJ, Wilson KS, Henrissat B (1997) Nomenclature for sugar-binding subsites in glycosyl hydrolases. Biochem J 321:557–559

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Dogra V, Sreenivasulu Y (2015) Cloning and functional characterization of β-1,3-glucanase gene from Podophyllum hexandrum - a high altitude Himalayan plant. Gene 554:25–31

    Article  CAS  PubMed  Google Scholar 

  • Douglas CM (2001) Fungal β(1,3)-D-glucan synthesis. Med Mycol 39:55–66

    Article  CAS  PubMed  Google Scholar 

  • Driskill LE, Bauer MW, Kelly RM (1999) Synergistic interactions among β-laminarinase, β-1,4-glucanase, and β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus during hydrolysis of β-1,4-, β-1,3-, and mixed-linked polysaccharides. Biotechnol Bioeng 66:51–60

    Article  CAS  PubMed  Google Scholar 

  • Feng HY, Drone J, Hoffmann L, Tran V, Tellier C, Rabiller C, Dion M (2005) Converting a β-glycosidase into a β-transglycosidase by directed evolution. J Biol Chem 280:37088–37097

    Article  CAS  PubMed  Google Scholar 

  • Fibriansah G, Masuda S, Koizumi N, Nakamura S, Kumasaka T (2007) The 1.3 Å crystal structure of a novel endo-β-1,3-glucanase of glycoside hydrolase family 16 from alkaliphilic Nocardiopsis sp. Strain F96. Proteins 69:683–690

    Article  CAS  PubMed  Google Scholar 

  • Fontaine T, Simenel C, Dubreucq G, Adam O, Delepierre M, Lemoine J, Vorgias CE, Diaquin M, Latge JP (2000) Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall. J Biol Chem 275:27594–27607

    CAS  PubMed  Google Scholar 

  • Gastebois A, Mouyna I, Simenel C, Clavaud C, Coddeville B, Delepierre M, Latge JP, Fontaine T (2010) Characterization of a new β(1–3)-glucan branching activity of Aspergillus fumigatus. J Biol Chem 285:2386–2396

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Goldman RC, Sullivan PA, Zakula D, Capobianco JO (1995) Kinetics of β-1,3 glucan interaction at the donor and acceptor sites of the fungal glucosyltransferase encoded by the BGL2 gene. FEBS J 227:372–378

    Article  CAS  Google Scholar 

  • Gueguen Y, Voorhorst WGB, van der Oost J, de Vos WM (1997) Molecular and biochemical characterization of an endo-β-1,3-glucanase of the hyperthermophilic archaeon Pyrococcus furiosus. J Biol Chem 272:31258–31264

    Article  CAS  PubMed  Google Scholar 

  • Guo Y, Yan Q, Yang Y, Yang S, Liu Y, Jiang Z (2015) Expression and characterization of a novel β-glucosidase, with transglycosylation and exo-β-1,3-glucanase activities, from Rhizomucor miehei. Food Chem 175:431–438

    Article  CAS  PubMed  Google Scholar 

  • Hoj PB, Slade AM, Wettenhall REH, Fincher GB (1988) Isolation and characterization of a (1 → 3)-β-glucan endohydrolase from germinating barley (Hordeum vulgare) - amino-acid sequence similarity with barley (1 → 3, 1 → 4)-β-glucanases. FEBS Lett 230:67–71

    Article  CAS  Google Scholar 

  • Hong TY, Cheng CW, Huang JW, Meng MS (2002) Isolation and biochemical characterization of an endo-1,3-β-glucanase from Streptomyces sioyaensis containing a C-terminal family 6 carbohydrate-binding module that binds to 1,3-β-glucan. Microbiology 148:1151–1159

    Article  CAS  PubMed  Google Scholar 

  • Hrmova M, Fincher GB (1993) Purification and properties of three (1 → 3)-β-D-glucanase isoenzymes from young leaves of barley (Hordeum vulgare). Biochem J 289:453–461

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Jiang L, Mishra P, Hietpas RT, Zeldovich KB, Bolon DN (2013) Latent effects of Hsp90 mutants revealed at reduced expression levels. PLoS Genet 9:e1003600

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Katrolia P, Jia H, Yan Q, Song S, Jiang Z, Xu H (2012) Characterization of a protease-resistant α-galactosidase from the thermophilic fungus Rhizomucor miehei and its application in removal of raffinose family oligosaccharides. Bioresour Technol 110:578–586

    Article  CAS  PubMed  Google Scholar 

  • Keen NT, Yoshikawa M (1983) β-1,3-endoglucanase from soybean releases elicitor active carbohydrates from fungus cell-walls. Plant Physiol 71:460–465

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kuroki R, Weaver LH, Matthews BW (1999) Structural basis of the conversion of T4 lysozyme into a transglycosidase by reengineering the active site. Proc Natl Acad Sci U S A 96:8949–8954

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–495

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  • Martin-Cuadrado AB, Fontaine T, Esteban PF, del Dedo JE, de Medina-Redondo M, del Rey F, Latge JP, de Aldana CR (2008) Characterization of the endo-β-1,3-glucanase activity of S. cerevisiae Eng2 and other members of the GH81 family. Fungal Genet Biol 45:542–553

    Article  CAS  PubMed  Google Scholar 

  • Mouyna I, Hartland RP, Fontaine T, Diaquin M, Simenel C, Delepierre M, Henrissat B, Latge JP (1998) A 1,3-β-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus is a homologue of the yeast Bgl2p. Microbiology 144:3171–3180

    Article  CAS  PubMed  Google Scholar 

  • Mouyna I, Fontaine T, Vai M, Monod M, Fonzi WA, Diaquin M, Popolo L, Hartland RP, Latge JP (2000) Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. J Biol Chem 275:14882–14889

    Article  CAS  PubMed  Google Scholar 

  • Mouyna I, Hartl L, Latge JP (2013) β-1,3-glucan modifying enzymes in Aspergillus fumigatus. Front Microbiol 4:81

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Notredame C, Higgins DG, Heringa J (2000) T-coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302:205–217

    Article  CAS  PubMed  Google Scholar 

  • Peumans WJ, Barre A, Derycke V, Rouge P, Zhang W, May GD, Delcour JA, Van Leuven F, Van Damme EJ (2000) Purification, characterization and structural analysis of an abundant β-1,3-glucanase from banana fruit. FEBS J 267:1188–1195

    Article  CAS  Google Scholar 

  • Qiao LX, Ding X, Wang HC, Sui JM, Wang JS (2014) Characterization of the β-1,3-glucanase gene in peanut (Arachis hypogaea L.) by cloning and genetic transformation. Genet Mol Res 13:1893–1904

    Article  CAS  PubMed  Google Scholar 

  • Qin Z, Yan Q, Lei J, Yang S, Jiang Z, Wu S (2015) The first crystal structure of a glycoside hydrolase family 17 β-1,3-glucanosyltransferase displays a unique catalytic cleft. Acta Crystallogr D Biol Crystallogr 71:1714–1724

    Article  CAS  PubMed  Google Scholar 

  • Rodriguez-Romero A, Hernandez-Santoyo A, Fuentes-Silva D, Palomares LA, Munoz-Cruz S, Yepez-Mulia L, Orozco-Martinez S (2014) Structural analysis of the endogenous glycoallergen Hev b 2 (endo-β-1,3-glucanase) from Hevea brasiliensis and its recognition by human basophils. Acta Crystallogr D Biol Crystallogr 70:329–341

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sakamoto Y, Nakade K, Konno N (2011) Endo-β-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family. Appl Environ Microbiol 77:8350–8354

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sarthy AV, McGonigal T, Coen M, Frost DJ, Meulbroek JA, Goldman RC (1997) Phenotype in Candida albicans of a disruption of the BGL2 gene encoding a 1,3-β-glucosyltransferase. Microbiology 143:367–376

    Article  CAS  PubMed  Google Scholar 

  • Sestak S, Hagen I, Tanner W, Strahl S (2004) Scw10p, a cell-wall glucanase/transglucosidase important for cell-wall stability in Saccharomyces cerevisiae. Microbiology 150:3197–3208

    Article  CAS  PubMed  Google Scholar 

  • Sokalingam S, Raghunathan G, Soundrarajan N, Lee SG (2012) A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein. PLoS One 7:e40410

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Teze D, Hendrickx J, Czjzek M, Ropartz D, Sanejouand YH, Tran V, Tellier C, Dion M (2014) Semi-rational approach for converting a GH1 β-glycosidase into a β-transglycosidase. Protein Eng Des Sel 27:13–19

    Article  CAS  PubMed  Google Scholar 

  • Varghese JN, Garrett TP, Colman PM, Chen L, Hoj PB, Fincher GB (1994) Three-dimensional structures of two plant β-glucan endohydrolases with distinct substrate specificities. Proc Natl Acad Sci U S A 91:2785–2789

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wojtkowiak A, Witek K, Hennig J, Jaskolski M (2013) Structures of an active-site mutant of a plant 1,3-β-glucanase in complex with oligosaccharide products of hydrolysis. Acta Crystallogr D Biol Crystallogr 69:52–62

    Article  CAS  PubMed  Google Scholar 

  • Woo JB, Kang HN, Woo EJ, Lee SB (2014) Molecular cloning and functional characterization of an endo-β-1,3-glucanase from Streptomyces matensis ATCC 23935. Food Chem 148:184–187

    Article  CAS  PubMed  Google Scholar 

  • Wu Y, Yuan S, Chen S, Wu D, Chen J, Wu J (2013) Enhancing the production of galacto-oligosaccharides by mutagenesis of Sulfolobus solfataricus β-galactosidase. Food Chem 138:1588–1595

    Article  CAS  PubMed  Google Scholar 

  • Zhou P, Liu Y, Yan Q, Chen Z, Qin Z, Jiang Z (2014) Structural insights into the substrate specificity and transglycosylation activity of a fungal glycoside hydrolase family 5 β-mannosidase. Acta Crystallogr D Biol Crystallogr 70:2970–2982

    Article  CAS  PubMed  Google Scholar 

  • Zverlov VV, Volkov IY, Velikodvorskaya TV, Schwarz WH (1997) Highly thermostable endo-1,3-β-glucanase (laminarinase) LamA from Thermotoga neapolitana: nucleotide sequence of the gene and characterization of the recombinant gene product. Microbiology 143:1701–1708

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported in part by the National Natural Science Foundation of China (No. 31471688), National Science Fund for Distinguished Young Scholars (No. 31325021), and Program for Changjiang Scholars (No. T2014055).

Author information

Authors and Affiliations

  1. College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, No.17 Qinghua Donglu, Haidian District, Post Box 294, Beijing, 100083, China

    Zhen Qin, Shaoqing Yang & Zhengqiang Jiang

  2. Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, No.17 Qinghua Donglu, Haidian District, Post Box 294, Beijing, 100083, China

    Qiaojuan Yan

Authors
  1. Zhen Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qiaojuan Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shaoqing Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhengqiang Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Qiaojuan Yan or Zhengqiang Jiang.

Ethics declarations

The work described has not been submitted elsewhere for publication, in whole or in part, and all authors listed carry out the data analysis and manuscript writing. This article does not contain any studies with animals performed by any of the authors.

Conflict of interest

The authors declare that they have no direct or indirect conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 244 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Qin, Z., Yan, Q., Yang, S. et al. Modulating the function of a β-1,3-glucanosyltransferase to that of an endo-β-1,3-glucanase by structure-based protein engineering. Appl Microbiol Biotechnol 100, 1765–1776 (2016). https://doi.org/10.1007/s00253-015-7057-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-015-7057-4

Keywords

Search

Navigation