tailieunhanh - Báo cáo khoa học: The absence of an identifiable single catalytic base residue in Thermobifida fusca exocellulase Cel6B

Thermobifida fuscaexocellulase Cel6B acts by an inverting hydrolysis mech-anism; however, the catalytic acid and base residues for this enzyme have not been confirmed. Site-directed mutagenesis and kinetic studies were used to show that Asp274 is the catalytic acid, which is consistent with what is found for other members of family-6 glycoside hydrolases; however, a sin-gle catalytic base was not identified. | ỊFEBS Journal The absence of an identifiable single catalytic base residue in Thermobifida fusca exocellulase Cel6B Thu V. Vuong and David B. Wilson Department of Molecular Biology and Genetics CornellUniversity Ithaca NY USA Keywords catalytic base exocellulase glycoside hydrolase hydrolysis mechanism proton-transferring network Correspondence D. B. Wilson Department of Molecular Biology and Genetics CornellUniversity Ithaca NY 14853 USA Fax 1 607 255 2428 Tel 1 607 255 5706 E-mail dbw3@ Received 7 April2009 revised 12 May 2009 accepted 14 May 2009 Thermobifida fusca exocellulase Cel6B acts by an inverting hydrolysis mechanism however the catalytic acid and base residues for this enzyme have not been confirmed. Site-directed mutagenesis and kinetic studies were used to show that Asp274 is the catalytic acid which is consistent with what is found for other members of family-6 glycoside hydrolases however a single catalytic base was not identified. Mutation of all putative catalytic base residues within 6 A of the -1 1 glucose subsites including the highly conserved Asp226 Asp497 and Glu495 as well as Ser232 and Tyr220 did not reveal a catalytic base although these residues are all important for activity. We propose a novel hydrolysis mechanism for T. fusca Cel6B involving a proton-transferring network to carry out the catalytic base function. doi Introduction Thermobifida fusca is an aerobic filamentous soil bacterium Actinomycetaceae that utilizes cellulose as its sole carbon source by producing a mixture of functionally distinct cellulases which act synergistically. Among them Cel6B is very important for achieving the maximum activity of synergistic mixtures although its activity alone is relatively weak on all polysaccharide substrates 1 . T. fusca Cel6B TfCel6B is an exocellulase EC that processively hydrolyzes b-1 4-glycosidic bonds from the nonreducing end of cellulose molecules. The enzyme has higher .