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. 1999 Apr 1;339(Pt 1):43–53.

Lignocellulose degradation by Phanerochaete chrysosporium: purification and characterization of the main alpha-galactosidase.

H Brumer 3rd 1, P F Sims 1, M L Sinnott 1
PMCID: PMC1220126  PMID: 10085226

Abstract

The main alpha-galactosidase was purified to homogeneity, in 30% yield, from a solid culture of Phanerochaete chrysosporium on 1 part wheat bran/2 parts thermomechanical softwood pulp. It is a glycosylated tetramer of 50 kDa peptide chains, which gives the N-terminal sequence ADNGLAITPQMG(?W)NT(?W)NHFG(?W)DIS(?W)DTI. It is remarkably stable, with crude extracts losing no activity over 3 h at 80 degrees C, and the purified enzyme retaining its activity over several months at 4 degrees C. The kinetics of hydrolysis at 25 degrees C of various substrates by this retaining enzyme were measured, absolute parameters being obtained by active-site titration with 2',4',6'-trinitrophenyl 2-deoxy-2, 2-difluoro-alpha-D-galactopyranoside. The variation of kcat/Km for 1-naphthyl-alpha-D-galactopyranoside with pH is bell-shaped, with pK1=1.91 and pK2=5.54. The alphaD(V/K) value for p-nitrophenyl-alpha-D-glucopyranoside is 1.031+/-0.007 at the optimal pH of 3.75 and 1.114+/-0.006 at pH7.00, indicating masking of the intrinsic effect at optimal pH. There is no alpha-2H effect on binding galactose [alphaD(Ki)=0.994+/-0.013]. The enzyme hydrolyses p-nitrophenyl beta-L-arabinopyranoside approximately 510 times slower than the galactoside, but has no detectable activity on the alpha-D-glucopyranoside or alpha-D-mannopyranoside. Hydrolysis of alpha-galactosides with poor leaving groups is Michaelian, but that of substrates with good leaving groups exhibits pronounced apparent substrate inhibition, with Kis values similar to Km values. We attribute this to the binding of the second substrate molecule to a beta-galactopyranosyl-enzyme intermediate, forming an E.betaGal. alphaGalX complex which turns over slowly, if at all. 1-Fluoro-alpha-D-galactopyranosyl fluoride, unlike alpha-D-galactopyranosyl fluoride, is a Michaelian substrate, indicating that the effect of 1-fluorine substitution is greater on the first than on the second step of the enzyme reaction.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. 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 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Braun C., Brayer G. D., Withers S. G. Mechanism-based inhibition of yeast alpha-glucosidase and human pancreatic alpha-amylase by a new class of inhibitors. 2-Deoxy-2,2-difluoro-alpha-glycosides. J Biol Chem. 1995 Nov 10;270(45):26778–26781. doi: 10.1074/jbc.270.45.26778. [DOI] [PubMed] [Google Scholar]
  4. Broda P., Birch P. R., Brooks P. R., Sims P. F. Lignocellulose degradation by Phanerochaete chrysosporium: gene families and gene expression for a complex process. Mol Microbiol. 1996 Mar;19(5):923–932. doi: 10.1046/j.1365-2958.1996.474966.x. [DOI] [PubMed] [Google Scholar]
  5. Broda P., Birch P., Brooks P., Copa-Patiño J. L., Sinnott M. L., Tempelaars C., Wang Q., Wyatt A., Sims P. Phanerochaete chrysosporium and its natural substrate. FEMS Microbiol Rev. 1994 Mar;13(2-3):189–195. doi: 10.1111/j.1574-6976.1994.tb00042.x. [DOI] [PubMed] [Google Scholar]
  6. Castanares A., Hay A. J., Gordon A. H., McCrae S. I., Wood T. M. D-xylan-degrading enzyme system from the fungus Phanerochaete chrysosporium: isolation and partial characterisation of an alpha-(4-O-methyl)-D-glucuronidase. J Biotechnol. 1995 Dec 15;43(3):183–194. doi: 10.1016/0168-1656(95)00128-x. [DOI] [PubMed] [Google Scholar]
  7. Clarke J. H., Rixon J. E., Ciruela A., Gilbert H. J., Hazlewood G. P. Family-10 and family-11 xylanases differ in their capacity to enhance the bleachability of hardwood and softwood paper pulps. Appl Microbiol Biotechnol. 1997 Aug;48(2):177–183. doi: 10.1007/s002530051035. [DOI] [PubMed] [Google Scholar]
  8. Copa-Patiño J. L., Broda P. A Phanerochaete chrysosporium beta-D-glucosidase/beta-D-xylosidase with specificity for (1-->3)-beta-D-glucan linkages. Carbohydr Res. 1994 Feb 3;253:265–275. doi: 10.1016/0008-6215(94)80071-5. [DOI] [PubMed] [Google Scholar]
  9. Covert S. F., Vanden Wymelenberg A., Cullen D. Structure, organization, and transcription of a cellobiohydrolase gene cluster from Phanerochaete chrysosporium. Appl Environ Microbiol. 1992 Jul;58(7):2168–2175. doi: 10.1128/aem.58.7.2168-2175.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dale M. P., Kopfler W. P., Chait I., Byers L. D. Beta-glucosidase: substrate, solvent, and viscosity variation as probes of the rate-limiting steps. Biochemistry. 1986 May 6;25(9):2522–2529. doi: 10.1021/bi00357a036. [DOI] [PubMed] [Google Scholar]
  11. Gold M. H., Alic M. Molecular biology of the lignin-degrading basidiomycete Phanerochaete chrysosporium. Microbiol Rev. 1993 Sep;57(3):605–622. doi: 10.1128/mr.57.3.605-622.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Henrissat B., Bairoch A. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1993 Aug 1;293(Pt 3):781–788. doi: 10.1042/bj2930781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Kachurin A. M., Golubev A. M., Geisow M. M., Veselkina O. S., Isaeva-Ivanova L. S., Neustroev K. N. Role of methionine in the active site of alpha-galactosidase from Trichoderma reesei. Biochem J. 1995 Jun 15;308(Pt 3):955–964. doi: 10.1042/bj3080955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kelly M. A., Sinnott M. L., Herrchen M. Purification and mechanistic properties of an extracellular alpha-L-arabinofuranosidase from Monilinia fructigena. Biochem J. 1987 Aug 1;245(3):843–849. doi: 10.1042/bj2450843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Konstantinidis A., Sinnott M. L. The interaction of 1-fluoro-D-glucopyranosyl fluoride with glucosidases. Biochem J. 1991 Oct 15;279(Pt 2):587–593. doi: 10.1042/bj2790587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Margolles-Clark E., Tenkanen M., Luonteri E., Penttilä M. Three alpha-galactosidase genes of Trichoderma reesei cloned by expression in yeast. Eur J Biochem. 1996 Aug 15;240(1):104–111. doi: 10.1111/j.1432-1033.1996.0104h.x. [DOI] [PubMed] [Google Scholar]
  19. McCarter J. D., Adam M. J., Braun C., Namchuk M., Tull D., Withers S. G. Syntheses of 2-deoxy-2-fluoro mono- and oligo-saccharide glycosides from glycals and evaluation as glycosidase inhibitors. Carbohydr Res. 1993 Oct 18;249(1):77–90. doi: 10.1016/0008-6215(93)84061-a. [DOI] [PubMed] [Google Scholar]
  20. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  21. Selwood T., Sinnott M. L. One-proton catalysis by the alpha-L-arabinofuranosidase III of Monilinia fructigena. Biochem J. 1988 Sep 15;254(3):899–901. doi: 10.1042/bj2540899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Srinivasan K., Konstantinidis A., Sinnott M. L., Hall B. G. Large changes of transition-state structure during experimental evolution of an enzyme. Biochem J. 1993 Apr 1;291(Pt 1):15–17. doi: 10.1042/bj2910015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Uzcategui E., Johansson G., Ek B., Pettersson G. The 1,4-beta-D-glucan glucanohydrolases from Phanerochaete chrysosporium. Re-assessment of their significance in cellulose degradation mechanisms. J Biotechnol. 1991 Nov;21(1-2):143–159. doi: 10.1016/0168-1656(91)90267-y. [DOI] [PubMed] [Google Scholar]
  24. Zeilinger S., Kristufek D., Arisan-Atac I., Hodits R., Kubicek C. P. Conditions of formation, purification, and characterization of an alpha-galactosidase of Trichoderma reesei RUT C-30. Appl Environ Microbiol. 1993 May;59(5):1347–1353. doi: 10.1128/aem.59.5.1347-1353.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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