- Department of Molecular Biology, Swedish University of Agricultural Sciences
Wu, Miao; Nerinckx, W; Piens, Kathleen; Ishida, Takuya; Hansson, Henrik; Sandgren, Mats; Ståhlberg, Jerry
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.
cellobiohydrolase; cellulase; exo-anomeric effect; Hypocrea jecorina; methylumbelliferyl-b–cellobioside
2013, Volume: 280, number: 1, pages: 184-198
Biochemistry and Molecular Biology