title: Mutation of a conserved tryptophan residue in the CBM3c of a GH9 endoglucanase inhibits activity.
abstract: The presence of the family of 3c cellulose binding module (CBM3c) is important for the catalytic activity of family 9 endoglucanases such as the EngZ from Clostridium cellulovorans. To determine the role of CBM3c in catalytic activity, we made a tryptophan to alanine substitution because tryptophan can bind strongly to both substrates and other amino acids. The conserved tryptophan substitution (W483A) did not influence substrate binding, but it reduced enzyme activity to 10-14% on both amorphous and crystalline cellulose. CBM3c is directly involved in the endoglucanase reaction independent of substrate binding. EngZ W483A was also inactivated independent of substrate concentrations. Specially, EngZ W483A restored its catalytic base activity (31.6±1.2U/nM) which is similar to the wild-type (29.4±0.3U/nM) on Avicel in the presence of 50mM sodium azide which is instead of catalytic base reaction. These results suggest that CBM3c is deeply involved in the cellulolytic reaction, specifically at the catalytic base region. Moreover, EngZ W483A was also easily denatured by DTT, an outer disulfide bond breaker, compared to the wild-type. CBM3c could influence the surface stability. These features of CBM3c result from the hydrophobic interaction of tryptophan with the catalytic domain that is unrelated to substrate binding.
authors.affiliation: Department of Biotechnology, Korea University, Seoul 02841, Republic of Korea.
authors.forename: Su-Jung
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, Korea University, Seoul 02841, Republic of Korea.
authors.forename: So Hyeong
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, Korea University, Seoul 02841, Republic of Korea.
authors.forename: Sang Kyu
authors.initials: SK
authors.lastname: Shin
authors.affiliation: Institute of Life Science and Natural Resources, Korea University, Seoul 02841, Republic of Korea.
authors.forename: Jeong Eun
authors.initials: JE
authors.lastname: Hyeon
authors.affiliation: Department of Biotechnology, Korea University, Seoul 02841, Republic of Korea; Institute of Life Science and Natural Resources, Korea University, Seoul 02841, Republic of Korea. Electronic address: samhan@korea.ac.kr.
authors.forename: Sung Ok
authors.initials: SO
authors.lastname: Han
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002480:Cellulase / Q000737:chemistry / Q000235:genetics; D046974:Clostridium cellulovorans / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D014364:Tryptophan; D002480:Cellulase
keywords: CD and CBM interaction; Catalytic base; Cellulose binding module 3c; Disulfide bond; Family 9 endoglucanase; Initial cellulolytic reaction