Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Jan 15;353(Pt 2):199–205. doi: 10.1042/0264-6021:3530199

Identification of essential active-site residues in the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis.

Z Keresztessy 1, K Brown 1, M A Dunn 1, M A Hughes 1
PMCID: PMC1221559  PMID: 11139381

Abstract

The coding sequence of the mature cyanogenic beta-glucosidase (beta-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin (K(m)=1.06 mM) and the synthetic p-nitrophenyl beta-D-glucopyranoside (PNP-Glc; K(m)=0.36 mM), as well as apparent pK(a) values of the free enzyme and the enzyme-substrate complexes (pK(E)(1)=4.4-4.8, pK(E)(2)=6.7-7.2, pK(ES)(1)=3.9-4.4, pK(ES)(2)=8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala-201Val mutation shifted the pK(a) of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased K(m) for linamarin hydrolysis 16-fold (16.1 mM) and that for PNP-Glc only 2.5-fold (0.84 mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava beta-glucosidase.

Full Text

The Full Text of this article is available as a PDF (175.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barrett T., Suresh C. G., Tolley S. P., Dodson E. J., Hughes M. A. The crystal structure of a cyanogenic beta-glucosidase from white clover, a family 1 glycosyl hydrolase. Structure. 1995 Sep 15;3(9):951–960. doi: 10.1016/s0969-2126(01)00229-5. [DOI] [PubMed] [Google Scholar]
  2. Burmeister W. P., Cottaz S., Driguez H., Iori R., Palmieri S., Henrissat B. The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an S-glycosidase. Structure. 1997 May 15;5(5):663–675. doi: 10.1016/s0969-2126(97)00221-9. [DOI] [PubMed] [Google Scholar]
  3. Carlson M., Taussig R., Kustu S., Botstein D. The secreted form of invertase in Saccharomyces cerevisiae is synthesized from mRNA encoding a signal sequence. Mol Cell Biol. 1983 Mar;3(3):439–447. doi: 10.1128/mcb.3.3.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chi Y. I., Martinez-Cruz L. A., Jancarik J., Swanson R. V., Robertson D. E., Kim S. H. Crystal structure of the beta-glycosidase from the hyperthermophile Thermosphaera aggregans: insights into its activity and thermostability. FEBS Lett. 1999 Feb 26;445(2-3):375–383. doi: 10.1016/s0014-5793(99)00090-3. [DOI] [PubMed] [Google Scholar]
  5. Cortés M. L., de Felipe P., Martín V., Hughes M. A., Izquierdo M. Successful use of a plant gene in the treatment of cancer in vivo. Gene Ther. 1998 Nov;5(11):1499–1507. doi: 10.1038/sj.gt.3300751. [DOI] [PubMed] [Google Scholar]
  6. Davies G., Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure. 1995 Sep 15;3(9):853–859. doi: 10.1016/S0969-2126(01)00220-9. [DOI] [PubMed] [Google Scholar]
  7. Hakulinen N., Paavilainen S., Korpela T., Rouvinen J. The crystal structure of beta-glucosidase from Bacillus circulans sp. alkalophilus: ability to form long polymeric assemblies. J Struct Biol. 2000 Feb;129(1):69–79. doi: 10.1006/jsbi.1999.4206. [DOI] [PubMed] [Google Scholar]
  8. Henrissat B., Bairoch A. Updating the sequence-based classification of glycosyl hydrolases. Biochem J. 1996 Jun 1;316(Pt 2):695–696. doi: 10.1042/bj3160695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hernández T., Lundquist P., Oliveira L., Pérez Cristiá R., Rodriguez E., Rosling H. Fate in humans of dietary intake of cyanogenic glycosides from roots of sweet cassava consumed in Cuba. Nat Toxins. 1995;3(2):114–117. doi: 10.1002/nt.2620030210. [DOI] [PubMed] [Google Scholar]
  10. Hughes J., Carvalho F. J., Hughes M. A. Purification, characterization, and cloning of alpha-hydroxynitrile lyase from cassava (Manihot esculenta Crantz). Arch Biochem Biophys. 1994 Jun;311(2):496–502. doi: 10.1006/abbi.1994.1267. [DOI] [PubMed] [Google Scholar]
  11. Hughes M. A., Brown K., Pancoro A., Murray B. S., Oxtoby E., Hughes J. A molecular and biochemical analysis of the structure of the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Cranz). Arch Biochem Biophys. 1992 Jun;295(2):273–279. doi: 10.1016/0003-9861(92)90518-2. [DOI] [PubMed] [Google Scholar]
  12. Keresztessy Z., Hughes J., Kiss L., Hughes M. A. Co-purification from Escherichia coli of a plant beta-glucosidase-glutathione S-transferase fusion protein and the bacterial chaperonin GroEL. Biochem J. 1996 Feb 15;314(Pt 1):41–47. doi: 10.1042/bj3140041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Keresztessy Z., Kiss L., Hughes M. A. Investigation of the active site of the cyanogenic beta-D-glucosidase (linamarase) from Manihot esculenta Crantz (cassava). I. Evidence for an essential carboxylate and a reactive histidine residue in a single catalytic center. Arch Biochem Biophys. 1994 Oct;314(1):142–152. doi: 10.1006/abbi.1994.1422. [DOI] [PubMed] [Google Scholar]
  14. Keresztessy Z., Kiss L., Hughes M. A. Investigation of the active site of the cyanogenic beta-D-glucosidase (linamarase) from Manihot esculenta Crantz (cassava). II. Identification of Glu-198 as an active site carboxylate group with acid catalytic function. Arch Biochem Biophys. 1994 Dec;315(2):323–330. doi: 10.1006/abbi.1994.1507. [DOI] [PubMed] [Google Scholar]
  15. Leary N. O., Pembroke A., Duggan P. F. Improving accuracy of glucose oxidase procedure for glucose determinations on discrete analyzers. Clin Chem. 1992 Feb;38(2):298–302. [PubMed] [Google Scholar]
  16. Ly H. D., Withers S. G. Mutagenesis of glycosidases. Annu Rev Biochem. 1999;68:487–522. doi: 10.1146/annurev.biochem.68.1.487. [DOI] [PubMed] [Google Scholar]
  17. McCarter J. D., Withers S. G. Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struct Biol. 1994 Dec;4(6):885–892. doi: 10.1016/0959-440x(94)90271-2. [DOI] [PubMed] [Google Scholar]
  18. Oxtoby E., Dunn M. A., Pancoro A., Hughes M. A. Nucleotide and derived amino acid sequence of the cyanogenic beta-glucosidase (linamarase) from white clover (Trifolium repens L.). Plant Mol Biol. 1991 Aug;17(2):209–219. doi: 10.1007/BF00039495. [DOI] [PubMed] [Google Scholar]
  19. Poulton J. E. Cyanogenesis in plants. Plant Physiol. 1990 Oct;94(2):401–405. doi: 10.1104/pp.94.2.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rudolph H. K., Antebi A., Fink G. R., Buckley C. M., Dorman T. E., LeVitre J., Davidow L. S., Mao J. I., Moir D. T. The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family. Cell. 1989 Jul 14;58(1):133–145. doi: 10.1016/0092-8674(89)90410-8. [DOI] [PubMed] [Google Scholar]
  21. Sali A., Blundell T. L. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993 Dec 5;234(3):779–815. doi: 10.1006/jmbi.1993.1626. [DOI] [PubMed] [Google Scholar]
  22. Sanz-Aparicio J., Hermoso J. A., Martínez-Ripoll M., Lequerica J. L., Polaina J. Crystal structure of beta-glucosidase A from Bacillus polymyxa: insights into the catalytic activity in family 1 glycosyl hydrolases. J Mol Biol. 1998 Jan 23;275(3):491–502. doi: 10.1006/jmbi.1997.1467. [DOI] [PubMed] [Google Scholar]
  23. Syrigos K. N., Rowlinson-Busza G., Epenetos A. A. In vitro cytotoxicity following specific activation of amygdalin by beta-glucosidase conjugated to a bladder cancer-associated monoclonal antibody. Int J Cancer. 1998 Dec 9;78(6):712–719. doi: 10.1002/(sici)1097-0215(19981209)78:6<712::aid-ijc8>3.0.co;2-d. [DOI] [PubMed] [Google Scholar]
  24. Tipton K. F., Dixon H. B. Effects of pH on enzymes. Methods Enzymol. 1979;63:183–234. doi: 10.1016/0076-6879(79)63011-2. [DOI] [PubMed] [Google Scholar]
  25. White A., Tull D., Johns K., Withers S. G., Rose D. R. Crystallographic observation of a covalent catalytic intermediate in a beta-glycosidase. Nat Struct Biol. 1996 Feb;3(2):149–154. doi: 10.1038/nsb0296-149. [DOI] [PubMed] [Google Scholar]
  26. Wiesmann C., Hengstenberg W., Schulz G. E. Crystal structures and mechanism of 6-phospho-beta-galactosidase from Lactococcus lactis. J Mol Biol. 1997 Jun 27;269(5):851–860. doi: 10.1006/jmbi.1997.1084. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES