Crystal Structure of Glycoside Hydrolase Family 9 Endoglucanase with an N-Terminal Ig-Like Domain Isolated from Leaf-Branch Compost

Abstract

Cellulase degrades glycosidic bonds of cellulose and is widely used for industrial purposes. Ten genes encoding novel cellulases, termed LC-CelA∼J, were isolated from leaf-branch compost by a metagenomic approach. Of them, LC-CelG, which is a member of glycoside hydrolase family 9 cellulases and contain an N-terminal immunoglobulin-like (Ig-like) domain, was overproduced in E. coli, purified, and characterized. LC-CelG consists of 577 amino acid residues. LC-CelG exhibited the highest activity at 70°C and pH 7.0. The crystal structure of LC-CelG was determined at 2.15A resolution. This structure resembles those of Clostridium thermocellum Cel9A (PDB: 1CLC), and contains two Ca2+ and one Zn2+ ions. Two conserved residues, Q40 and D99, in the Ig-like domain form hydrogen bonds and salt bridges respectively with the catalytic domain. To analyze the role of these interactions, two single (Q40A-CelG and D99A-CelG) and one double (Q40A/D99A-CelG) mutant proteins were constructed. The deletion mutant, ΔIg-CelG, which lacks the Ig-like domain, was also constructed. The far-UV CD spectra of these mutant proteins suggest that the structure of LC-CelG is not significantly changed by these mutations and deletion. Q40A-CelG and D99A-CelG were nearly as stable as LC-CelG, whereas Q40A/D99A-CelG and ΔIg-CelG were less stable than LC-CelG by 9.2 and 12.2°C respectively. This result suggests that Q40 and D99 cooperatively contribute to the stabilization of LC-CelG and removal of the Q40- and D99-mediated interactions is the main reason why LC-CelG is destabilized by deletion of the Ig-like domain. ΔIg-CelG was inactive at any temperature examined, whereas other mutants were as active as LC-CelG at ≤60°C, suggesting that the Ig-like domain is required for substrate binding or to make the conformation of the active site functional.