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Research article2013Peer reviewed

Rational design, synthesis, evaluation and enzyme-substrate structures of improved fluorogenic substrates for family 6 glycoside hydrolases

Wu, Miao; Nerinckx, W; Piens, Kathleen; Ishida, Takuya; Hansson, Henrik; Sandgren, Mats; Ståhlberg, Jerry

Abstract

Methylumbelliferyl-β–cellobioside (MUF–G2) is a convenient fluorogenic substrate for certain β-glycoside hydrolases (GH). However, hydrolysis of the aglycone is poor with GH family 6 enzymes (GH6), despite strong binding. Prediction of the orientation of the aglycone of MUF–G2 in the +1 subsite of Hypocrea jecorina Cel6A by automated docking suggested umbelliferyl modifications at C4 and C6 for improved recognition. Four modified umbelliferyl-β-cellobiosides [6-chloro-4-methyl-(ClMUF); 6-chloro-4-trifluoromethyl- (ClF3MUF); 4-phenyl- (PhUF); 6–chloro-4–phenyl- (ClPhUF)] were synthesized and tested with GH6, GH7, GH9, GH5 and GH45 cellulases. Indeed the rate of aglycone release by H. jecorina Cel6A was 10–150 times higher than with MUF-G2, although it was still three orders of magnitude lower than with H. jecorina Cel7B. The 4-phenyl substitution drastically reduced the fluorescence intensity of the free aglycone, while ClMUF-G2 could be used for determination of kcat and KM for H. jecorina Cel6A and Thermobifida fusca Cel6A. Crystal structures of H. jecorina Cel6A D221A mutant soaked with the MUF-, ClMUF- and ClPhUF-β-cellobioside substrates show that the modifications turned the umbelliferyl group ‘upside down’, with the glycosidic bond better positioned for protonation than with MUF-G2.

Keywords

cellobiohydrolase; cellulase; exo-anomeric effect; Hypocrea jecorina; methylumbelliferyl-b–cellobioside

Published in

FEBS Journal
2013, Volume: 280, number: 1, pages: 184-198

      SLU Authors

    • Wu, Miao

      • Department of Molecular Biology, Swedish University of Agricultural Sciences

        • Hansson, Henrik

          • Department of Molecular Biology, Swedish University of Agricultural Sciences

          • Sandgren, Mats

            • Department of Molecular Biology, Swedish University of Agricultural Sciences

            • Ståhlberg, Jerry

              • Department of Molecular Biology, Swedish University of Agricultural Sciences

            UKÄ Subject classification

            Structural Biology
            Biochemistry and Molecular Biology
            Organic Chemistry

            Publication identifier

            DOI: https://doi.org/10.1111/febs.12060

            Permanent link to this page (URI)

            https://res.slu.se/id/publ/39078