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

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 Cel6 A by automated docking suggested umbelliferyl modifications at C4 and C6 for improved recognition. Four modified umbelliferyl-β-cellobiosides [6-chloro-4-methyl- (Cl MUF); 6-chloro-4-trifluoromethyl- (ClF3 MUF); 4-phenyl- (Ph UF); 6-chloro-4-phenyl- (ClPh UF)] were synthesized and tested with GH6, GH7, GH9, GH5 and GH45 cellulases. Indeed the rate of aglycone release by H. jecorina Cel6 A was 10-150 times higher than with MUF- G2, although it was still three orders of magnitude lower than with H. jecorina Cel7 B. The 4-phenyl substitution drastically reduced the fluorescence intensity of the free aglycone, while Cl MUF- G2 could be used for determination of kcat and KM for H. jecorina Cel6 A and Thermobifida fusca Cel6 A. Crystal structures of H. jecorina Cel6 A D221 A mutant soaked with the MUF-, Cl MUF- and ClPh UF-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. Database Structural data have been submitted to the Protein Data Bank under accession numbers pdb 4AU0, 4AX7, 4AX6 Structured digital abstract • http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260296 • Cel6A and Cel6A bind by x-ray crystallography (View Interaction: 1, 2) [ABSTRACT FROM AUTHOR]