Date Published: July 01, 2018
Publisher: International Union of Crystallography
Author(s): Thomas L. Ellinghaus, Jose H. Pereira, Ryan P. McAndrew, Ditte H. Welner, Andy M. DeGiovanni, Joel M. Guenther, Huu M. Tran, Taya Feldman, Blake A. Simmons, Kenneth L. Sale, Paul D. Adams.
The engineering of metal binding into a cellulase increases its temperature stability while maintaining its other catalytic properties.
Concerns about climate, increasing energy demands and the limited long-term supply of fossil fuels, as well as ethical considerations, have promoted research on more sustainable lignocellulosic ‘second-generation’ biofuels. They rely on the deconstruction of cellulose and hemicellulose from feedstocks such as switchgrass into monosaccharides. One step in this process, saccharification, employs mixtures of thermally and chemically robust glycoside hydrolases (GHs). These mixtures typically make use of the combined activities of endoglucanases (EC 22.214.171.124), β-glucosidases (EC 126.96.36.199) and cellobiohydrolases (EC 188.8.131.52 and EC 184.108.40.206; CBHs). The Carbohydrate-Active enZymes database (CAZy; http://www.cazy.org) currently lists 153 different GH families based on amino-acid sequence similarities. This classification facilitates the grouping of GHs according to structural, mechanistic and evolutionary aspects independent of substrate specificities.
The positive effect of Ca2+ binding on the thermal tolerance of the cellulase CtCel9A has been shown previously, as has the correlation of a Zn2+-binding site with enzyme activity and structural integrity (Chauvaux et al., 1990 ▸, 1995 ▸). Our design of a mutant version of J30 based on structural data further underlines this relationship. Here, the majority of the increase in stability is likely to be a result of the net exchange of a hydrogen bond between the side chains of His115 and Tyr143 (J30 wt) for four coordinating bonds between the Zn2+ ion and the side chains of Cys98, Cys114, His115 and His143 (J30 CCH; Fig. 2 ▸). This stabilization may in part result from three separate sections of the polypeptide chain interacting via the Zn2+ ion. The structural and biochemical data strongly suggest that the metal-binding site is purely structural, yet it is also proximal to the active-site residues Asp144 and Asp147 and the region of the substrate cleft where the reducing end binds.