title: Rational design, synthesis, evaluation and enzyme-substrate structures of improved fluorogenic substrates for family 6 glycoside hydrolases.
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 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.
authors.affiliation: Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Miao
authors.initials: M
authors.lastname: Wu
authors.forename: Wim
authors.initials: W
authors.lastname: Nerinckx
authors.forename: Kathleen
authors.initials: K
authors.lastname: Piens
authors.forename: Takuya
authors.initials: T
authors.lastname: Ishida
authors.forename: Henrik
authors.initials: H
authors.lastname: Hansson
authors.forename: Mats
authors.initials: M
authors.lastname: Sandgren
authors.forename: Jerry
authors.initials: J
authors.lastname: Ståhlberg
mesh_terms: D000192:Actinomycetales / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry; D020134:Catalytic Domain; D002475:Cellobiose / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D044602:Cellulases / Q000737:chemistry*; D018360:Crystallography, X-Ray; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D006868:Hydrolysis; D035901:Hypocrea / Q000201:enzymology; D007700:Kinetics; D062105:Molecular Docking Simulation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D002475:Cellobiose; D044602:Cellulases