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. 2001 May 15;356(Pt 1):19–30. doi: 10.1042/0264-6021:3560019

Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/ A224H/L225V/T226A/D262G mutant.

D Becker 1, C Braet 1, H Brumer 3rd 1, M Claeyssens 1, C Divne 1, B R Fagerström 1, M Harris 1, T A Jones 1, G J Kleywegt 1, A Koivula 1, S Mahdi 1, K Piens 1, M L Sinnott 1, J Ståhlberg 1, T T Teeri 1, M Underwood 1, G Wohlfahrt 1
PMCID: PMC1221808  PMID: 11336632

Abstract

The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves. The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K(m) values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced non-productive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.

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Selected References

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  1. ALBERTY R. A., BLOOMFIELD V. MULTIPLE INTERMEDIATES IN STEADY STATE ENZYME KINETICS. V. EFFECT OF PH ON THE RATE OF A SIMPLE ENZYMATIC REACTION. J Biol Chem. 1963 Aug;238:2804–2810. [PubMed] [Google Scholar]
  2. Barr B. K., Hsieh Y. L., Ganem B., Wilson D. B. Identification of two functionally different classes of exocellulases. Biochemistry. 1996 Jan 16;35(2):586–592. doi: 10.1021/bi9520388. [DOI] [PubMed] [Google Scholar]
  3. Boisset C., Fraschini C., Schülein M., Henrissat B., Chanzy H. Imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A. Appl Environ Microbiol. 2000 Apr;66(4):1444–1452. doi: 10.1128/aem.66.4.1444-1452.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brünger A. T., Adams P. D., Clore G. M., DeLano W. L., Gros P., Grosse-Kunstleve R. W., Jiang J. S., Kuszewski J., Nilges M., Pannu N. S. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905–921. doi: 10.1107/s0907444998003254. [DOI] [PubMed] [Google Scholar]
  5. Béguin P., Lemaire M. The cellulosome: an exocellular, multiprotein complex specialized in cellulose degradation. Crit Rev Biochem Mol Biol. 1996 Jun;31(3):201–236. doi: 10.3109/10409239609106584. [DOI] [PubMed] [Google Scholar]
  6. Claeyssens M., Van Tilbeurgh H., Tomme P., Wood T. M., McRae S. I. Fungal cellulase systems. Comparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and Trichoderma reesei. Biochem J. 1989 Aug 1;261(3):819–825. doi: 10.1042/bj2610819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Divne C., Ståhlberg J., Reinikainen T., Ruohonen L., Pettersson G., Knowles J. K., Teeri T. T., Jones T. A. The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei. Science. 1994 Jul 22;265(5171):524–528. doi: 10.1126/science.8036495. [DOI] [PubMed] [Google Scholar]
  8. Divne C., Ståhlberg J., Teeri T. T., Jones T. A. High-resolution crystal structures reveal how a cellulose chain is bound in the 50 A long tunnel of cellobiohydrolase I from Trichoderma reesei. J Mol Biol. 1998 Jan 16;275(2):309–325. doi: 10.1006/jmbi.1997.1437. [DOI] [PubMed] [Google Scholar]
  9. Gilkes N. R., Jervis E., Henrissat B., Tekant B., Miller R. C., Jr, Warren R. A., Kilburn D. G. The adsorption of a bacterial cellulase and its two isolated domains to crystalline cellulose. J Biol Chem. 1992 Apr 5;267(10):6743–6749. [PubMed] [Google Scholar]
  10. 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]
  11. Henrissat B., Davies G. Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol. 1997 Oct;7(5):637–644. doi: 10.1016/s0959-440x(97)80072-3. [DOI] [PubMed] [Google Scholar]
  12. Henrissat B., Teeri T. T., Warren R. A. A scheme for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants. FEBS Lett. 1998 Mar 27;425(2):352–354. doi: 10.1016/s0014-5793(98)00265-8. [DOI] [PubMed] [Google Scholar]
  13. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  14. Joshi M. D., Sidhu G., Pot I., Brayer G. D., Withers S. G., McIntosh L. P. Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase. J Mol Biol. 2000 May 26;299(1):255–279. doi: 10.1006/jmbi.2000.3722. [DOI] [PubMed] [Google Scholar]
  15. Kleywegt G. J., Brünger A. T. Checking your imagination: applications of the free R value. Structure. 1996 Aug 15;4(8):897–904. doi: 10.1016/s0969-2126(96)00097-4. [DOI] [PubMed] [Google Scholar]
  16. Kleywegt G. J., Zou J. Y., Divne C., Davies G. J., Sinning I., Stâhlberg J., Reinikainen T., Srisodsuk M., Teeri T. T., Jones T. A. The crystal structure of the catalytic core domain of endoglucanase I from Trichoderma reesei at 3.6 A resolution, and a comparison with related enzymes. J Mol Biol. 1997 Sep 26;272(3):383–397. doi: 10.1006/jmbi.1997.1243. [DOI] [PubMed] [Google Scholar]
  17. Koivula A., Lappalainen A., Virtanen S., Mäntylä A. L., Suominen P., Teeri T. T. Immunoaffinity chromatographic purification of cellobiohydrolase II mutants from recombinant trichoderma reesei strains devoid of major endoglucanase genes. Protein Expr Purif. 1996 Dec;8(4):399–400. [PubMed] [Google Scholar]
  18. Lever M. A new reaction for colorimetric determination of carbohydrates. Anal Biochem. 1972 May;47(1):273–279. doi: 10.1016/0003-2697(72)90301-6. [DOI] [PubMed] [Google Scholar]
  19. MacKenzie L. F., Sulzenbacher G., Divne C., Jones T. A., Wöldike H. F., Schülein M., Withers S. G., Davies G. J. Crystal structure of the family 7 endoglucanase I (Cel7B) from Humicola insolens at 2.2 A resolution and identification of the catalytic nucleophile by trapping of the covalent glycosyl-enzyme intermediate. Biochem J. 1998 Oct 15;335(Pt 2):409–416. doi: 10.1042/bj3350409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mach R. L., Schindler M., Kubicek C. P. Transformation of Trichoderma reesei based on hygromycin B resistance using homologous expression signals. Curr Genet. 1994 Jun;25(6):567–570. doi: 10.1007/BF00351679. [DOI] [PubMed] [Google Scholar]
  21. Mackenzie L. F., Davies G. J., Schülein M., Withers S. G. Identification of the catalytic nucleophile of endoglucanase I from Fusarium oxysporum by mass spectrometry. Biochemistry. 1997 May 13;36(19):5893–5901. doi: 10.1021/bi962962h. [DOI] [PubMed] [Google Scholar]
  22. Margolles-Clark E., Hayes C. K., Harman G. E., Penttilä M. Improved production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei. Appl Environ Microbiol. 1996 Jun;62(6):2145–2151. doi: 10.1128/aem.62.6.2145-2151.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mock W. L., Stanford D. J. Arazoformyl dipeptide substrates for thermolysin. Confirmation of a reverse protonation catalytic mechanism. Biochemistry. 1996 Jun 11;35(23):7369–7377. doi: 10.1021/bi952827p. [DOI] [PubMed] [Google Scholar]
  24. Roberge M., Shareck F., Morosoli R., Kluepfel D., Dupont C. Site-directed mutagenesis study of a conserved residue in family 10 glycanases: histidine 86 of xylanase A from Streptomyces lividans. Protein Eng. 1998 May;11(5):399–404. doi: 10.1093/protein/11.5.399. [DOI] [PubMed] [Google Scholar]
  25. Srisodsuk M., Kleman-Leyer K., Keränen S., Kirk T. K., Teeri T. T. Modes of action on cotton and bacterial cellulose of a homologous endoglucanase-exoglucanase pair from Trichoderma reesei. Eur J Biochem. 1998 Feb 1;251(3):885–892. doi: 10.1046/j.1432-1327.1998.2510885.x. [DOI] [PubMed] [Google Scholar]
  26. Ståhlberg J., Divne C., Koivula A., Piens K., Claeyssens M., Teeri T. T., Jones T. A. Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolase I from Trichoderma reesei. J Mol Biol. 1996 Nov 29;264(2):337–349. doi: 10.1006/jmbi.1996.0644. [DOI] [PubMed] [Google Scholar]
  27. Sulzenbacher G., Driguez H., Henrissat B., Schülein M., Davies G. J. Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: substrate distortion gives rise to the preferred axial orientation for the leaving group. Biochemistry. 1996 Dec 3;35(48):15280–15287. doi: 10.1021/bi961946h. [DOI] [PubMed] [Google Scholar]
  28. Teleman A., Koivula A., Reinikainen T., Valkeajärvi A., Teeri T. T., Drakenberg T., Teleman O. Progress-curve analysis shows that glucose inhibits the cellotriose hydrolysis catalysed by cellobiohydrolase II from Trichoderma reesei. Eur J Biochem. 1995 Jul 1;231(1):250–258. doi: 10.1111/j.1432-1033.1995.tb20694.x. [DOI] [PubMed] [Google Scholar]
  29. Tomme P., Van Tilbeurgh H., Pettersson G., Van Damme J., Vandekerckhove J., Knowles J., Teeri T., Claeyssens M. Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis. Eur J Biochem. 1988 Jan 4;170(3):575–581. doi: 10.1111/j.1432-1033.1988.tb13736.x. [DOI] [PubMed] [Google Scholar]

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