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Rational design, synthesis, evaluation and enzyme – substrate structures of improved fluorogenic substrates for family 6 glycoside hydrolases
Author(s) -
Wu Miao,
Nerinckx Wim,
Piens Kathleen,
Ishida Takuya,
Hansson Henrik,
Sandgren Mats,
Ståhlberg Jerry
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12060
Subject(s) - aglycone , trisaccharide , chemistry , stereochemistry , glycosidic bond , substrate (aquarium) , glycoside , glycoside hydrolase , hypocrea , hydrolysis , cellulase , enzyme , biochemistry , trichoderma reesei , biology , ecology
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 k(cat) and K(M) 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.