Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A: A comparison with Phanerochaete chrysosporium Cel7D

Ingemar von Ossowski, Jerry Ståhlberg, Anu Koivula, Kathleen Piens, Dieter Becker, Harry Boer, Raija Harle, Mark Harris, Christina Divne, Sabah Mahdi, Yongxin Zhao, Hugues Driguez, Marc Claeyssens, Michael L. Sinnott, Tuula Teeri (Corresponding Author)

    Research output: Contribution to journalArticleScientificpeer-review

    145 Citations (Scopus)


    The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Δ(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations.

    The Y247F mutation caused a slight kcat reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both kcat and KM and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc2/(Glc3+Glc1) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.
    Original languageEnglish
    Pages (from-to)817-829
    Number of pages13
    JournalJournal of Molecular Biology
    Issue number4
    Publication statusPublished - 2003
    MoE publication typeA1 Journal article-refereed


    • cellulose
    • crystal structure
    • glycoside hydrolase
    • processivity
    • product inhibition


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