4AU0

Hypocrea jecorina Cel6A D221A mutant soaked with 6-chloro-4- methylumbelliferyl-beta-cellobioside

Summary for 4AU0

• Entry DOI: 10.2210/pdb4au0/pdb
• Related: 1CB2 1HGW 1HGY 1QJW 1QK0 1QK2 3CBH 4AX6 4AX7
• Related PRD ID: PRD_900005
• Descriptor: EXOGLUCANASE 2, beta-D-glucopyranose-(1-4)-beta-D-glucopyranose, 2-acetamido-2-deoxy-beta-D-glucopyranose, ... (7 entities in total)
• Functional Keywords: hydrolase, hydrolase(o-glycosyl), glycosidase, glycoside hydrolase, gh6, clmufg2, cellulase, glycoprotein, fluorogenic substrate
• Biological source: HYPOCREA JECORINA
• Cellular location: Secreted: P07987
• Total number of polymer chains: 2
• Total formula weight: 82607.45

Authors

Wu, M.,Nerinckx, W.,Piens, K.,Ishida, T.,Hansson, H.,Stahlberg, J.,Sandgren, M. (deposition date: 2012-05-11, release date: 2013-01-23, Last modification date: 2020-07-29)

Primary citation

Wu, M.,Nerinckx, W.,Piens, K.,Ishida, T.,Hansson, H.,Sandgren, M.,Stahlberg, J.
Rational Design, Synthesis, Evaluation and Enzyme-Substrate Structures of Improved Fluorogenic Substrates for Family 6 Glycoside Hydrolases.
FEBS J., 280:184-, 2013

PubMed 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.

PubMed: 23137336
DOI: 10.1111/FEBS.12060

Experimental method

X-RAY DIFFRACTION (1.7 Å)