Crystal structure and substrate‐binding mode of cellulase 12A from Thermotoga maritima

Abstract

Cellulases have been used in many applications to treat various carbohydrate-containing materials. Thermotoga maritima cellulase 12A (TmCel12A) belongs to the GH12 family of glycoside hydrolases. It is a β-1,4-endoglucanase that degrades cellulose molecules into smaller fragments, facilitating further utilization of the carbohydrate. Because of its hyperthermophilic nature, the enzyme is especially suitable for industrial applications. Here the crystal structure of TmCel12A was determined by using an active-site mutant E134C and its mercury-containing derivatives. It adopts a β-jellyroll protein fold typical of the GH12-family enzymes, with two curved β-sheets A and B and a central active-site cleft. Structural comparison with other GH12 enzymes shows significant differences, as found in two longer and highly twisted β-strands B8 and B9 and several loops. A unique Loop A3-B3 that contains Arg60 and Tyr61 stabilizes the substrate by hydrogen bonding and stacking, as observed in the complex crystals with cellotetraose and cellobiose. The high-resolution structures allow clear elucidation of the network of interactions between the enzyme and its substrate. The sugar residues bound to the enzyme appear to be more ordered in the -2 and -1 subsites than in the +1, +2 and -3 subsites. In the E134C crystals the bound -1 sugar at the cleavage site consistently show the α-anomeric configuration, implicating an intermediate-like structure.

Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop

“…The TmCel12A pET16b plasmid was constructed as described previously (Cheng et al 2011). Each mutant was prepared by using QuickChange site-directed mutagenesis kit (Agilent) with TmCel12A pET16b as the template.…”

“…Even so, few industrial cellulases with both high thermostability and high enzyme activity have been obtained. In our previous study of the crystal structures of T. maritima cellulase 12A (TmCel12A) and its complex with oligosaccharide, a unique surface loop between the strands A3 and B3 was found to protrude over the active-site cleft, forming an enclosure around the −1 sugar of the substrate (Cheng et al 2011). This loop, containing Arg60 and Tyr61, is longer than its equivalents in the other GH12-family enzymes and deviates significantly from them in sequence and structure.…”

Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-β-d-glucanase

“…The first is to directly clone the enzyme-coding genes from hyperthermophiles and to express the proteins in industrial strains. For instance, our recently solved Thermotoga maritima cellulase 12A (TmCel12A) X-ray structure that belongs to the GH12 family of glycoside hydrolases shows the strongest activity at 95°C and has a pH optimum of 5 (Bronnenmeier et al 1995;Liebl et al 1996;Cheng et al 2011). These characteristics make the enzyme highly valuable in various utilizations, since industrial processes such as plant waste treatments usually involve high temperature and low pH.…”

Structure and Catalytic Mechanism of a Glycoside Hydrolase Family-127 β-L-Arabinofuranosidase (HypBA1)

“…In our previous studies, mercury can bind to free Cys residues very easily and the phase problem can be easily solved by using at least two mercury datasets by MIR [18][19][20][21]. The mercury atom binding sites were located using AutoSol wizard of PHENIX using 2.4 Å resolution cutoff [22].…”