title: Structure and stability of metagenome-derived glycoside hydrolase family 12 cellulase (LC-CelA) a homolog of Cel12A from Rhodothermus marinus.
abstract: Ten genes encoding novel cellulases with putative signal peptides at the N-terminus, termed pre-LC-CelA-J, were isolated from a fosmid library of a leaf-branch compost metagenome by functional screening using agar plates containing carboxymethyl cellulose and trypan blue. All the cellulases except pre-LC-CelG have a 14-29 residue long flexible linker (FL) between the signal peptide and the catalytic domain. LC-CelA without a signal peptide (residues 20-261), which shows 76% amino acid sequence identity to Cel12A from Rhodothermus marinus (RmCel12A), was overproduced in Escherichia coli, purified and characterized. LC-CelA exhibited its highest activity across a broad pH range (pH 5-9) and at 90 °C, indicating that LC-CelA is a highly thermostable cellulase, like RmCel12A. The crystal structure of LC-CelA was determined at 1.85 Å resolution and is nearly identical to that of RmCel12A determined in a form without the FL. Both proteins contain two disulfide bonds. LC-CelA has a 16-residue FL (residues 20-35), most of which is not visible in the electron density map, probably due to structural disorder. However, Glu34 and Pro35 form hydrogen bonds with the central region of the protein. ΔFL-LC-CelA (residues 36-261) and E34A-LC-CelA with a single Glu34 → Ala mutation were therefore constructed and characterized. ΔFL-LC-CelA and E34A-LC-CelA had lower melting temperatures (T m) than LC-CelA by 14.7 and 12.0 °C respectively. The T m of LC-CelA was also decreased by 28.0 °C in the presence of dithiothreitol. These results suggest that Glu34-mediated hydrogen bonds and the two disulfide bonds contribute to the stabilization of LC-CelA.
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.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Ozaki
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Eiko
authors.initials: E
authors.lastname: Kanaya
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Joong-Jae
authors.initials: JJ
authors.lastname: Kim
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan ; International College, Osaka University, 1-30 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Clement
authors.initials: C
authors.lastname: Angkawidjaja
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Koga
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
keywords: CD, circular dichroism; CM-cellulose, carboxymethyl cellulose; Crystal structure; DTT, dithiothreitol; FL, flexible linker; Flexible linker; GH family, glycoside hydrolase family; GdnHCl, guanidine hydrochloride; Glycoside hydrolase family 12 cellulase; LC-CelA, GH family 12 cellulase from leaf–branch compost; Leaf–branch compost; Metagenome; SP, signal peptide; Stability