title: Structure, activity, and stability of metagenome-derived glycoside hydrolase family 9 endoglucanase with an N-terminal Ig-like domain.
abstract: A metagenome-derived glycoside hydrolase family 9 enzyme with an N-terminal immunoglobulin-like (Ig-like) domain, leaf-branch compost (LC)-CelG, was characterized and its crystal structure was determined. LC-CelG did not hydrolyze p-nitrophenyl cellobioside but hydrolyzed CM-cellulose, indicating that it is endoglucanase. LC-CelG exhibited the highest activity at 70°C and >80% of the maximal activity at a broad pH range of 5-9. Its denaturation temperature was 81.4°C, indicating that LC-CelG is a thermostable enzyme. The structure of LC-CelG resembles those of CelD from Clostridium thermocellum (CtCelD), Cel9A from Alicyclobacillus acidocaldarius (AaCel9A), and cellobiohydrolase CbhA from C. thermocellum (CtCbhA), which show relatively low (29-31%) amino acid sequence identities to LC-CelG. Three acidic active site residues are conserved as Asp194, Asp197, and Glu558 in LC-CelG. Ten of the thirteen residues that form the substrate binding pocket of AaCel9A are conserved in LC-CelG. Removal of the Ig-like domain reduced the activity and stability of LC-CelG by 100-fold and 6.3°C, respectively. Removal of the Gln40- and Asp99-mediated interactions between the Ig-like and catalytic domains destabilized LC-CelG by 5.0°C without significantly affecting its activity. These results suggest that the Ig-like domain contributes to the stabilization of LC-CelG mainly due to the Gln40- and Asp99-mediated interactions. Because the LC-CelG derivative lacking the Ig-like domain accumulated in Escherichia coli cells mostly in an insoluble form and this derivative accumulated in a soluble form exhibited very weak activity, the Ig-like domain may be required to make the conformation of the active site functional and prevent aggregation of the catalytic domain.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Okano
authors.forename: Eiko
authors.initials: E
authors.lastname: Kanaya
authors.forename: Masashi
authors.initials: M
authors.lastname: Ozaki
authors.forename: Clement
authors.initials: C
authors.lastname: Angkawidjaja
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D007136:Immunoglobulins; D054892:Metagenome; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D007136:Immunoglobulins; D011994:Recombinant Proteins; D002480:Cellulase; D015032:Zinc
keywords: GH family 9; crystal structure; endoglucanase; immunoglobulin-like (Ig-like) domain; metagenome; thermal stability