Site-Directed Mutation of the Putative Catalytic Residues of Endoglucanase CenA from Cellulomonas fimi

Abstract

The catalytic domains of beta-1,4-glucanases can be grouped into families of related amino acid sequences. The endoglucanase CenA from Cellulomonas fimi is a member of family B. All enzymes from this family are believed to hydrolyze beta-1,4-glucosidic bonds using a general acid-base catalytic mechanism resulting in inversion of anomeric configuration at the scissile bond. Three-dimensional structures for two cellulases from family B have been determined by X-ray crystallographic analysis. These structures show that there are four Asp residues which are in a position to function as acid catalyst, base catalyst, and/or transition state stabilizers. These aspartates are conserved in all members of family B. The roles of Asp216, Asp252, Asp287, and Asp392, the corresponding amino acids in CenA, were determined. These aspartates have been systematically replaced with alanine and glutamate via site-directed mutagenesis, and the resulting effect on activity, substrate specificity, and overall structure has been determined. Changes in overall structure were monitored using circular dichroism spectroscopy, and no significant differences between the wild-type and mutant proteins were found. Active site structure was also found to be intact as all proteins bound to a cellobiose affinity column. The Michaelis-Menten parameters of the enzyme were determined on 2,4-dinitrophenyl cellobioside as well as (carboxymethyl)-cellulose and phosphoric acid-swollen cellulose. Initial characterization of mutant proteins indicates that Asp252 and Asp392 are the acid and base catalysts, respectively, in CenA. Residue Asp287 appears to aid Asp252 in acid catalysis, and Asp216 is not absolutely required for catalysis.

Advantages of a distant cellulase catalytic base

“…Calculations of low-energy conformations of the system in the hydrolysis-ready position with the −2 and −1 positions occupied by the cellulose chain indicate that the overall reaction barrier in the D175E mutant would be increased by at least between 1.5 and 2.5 kcal/mol. While this is a qualitative result, it corresponds with an activity reduction of approximately two orders of magnitude, and is therefore approximately consistent with measured activity reductions for the homologous mutants Tf Cel6A D79E (20) and Cf Cel6A D216E (21). This agreement allows us to address our primary question of why a longer side chain for the nucleophilic residue does not produce a more active enzyme.…”

“…This suggests investigation of a TrCel6A D175E mutant, since glutamate (E) is identical in structure to aspartate (D) but for an additional CH 2 group in its side chain that could project its carboxylate group further into the active site. While no studies appear in the literature on a TrCel6A D175E mutant, mutants of the homologous aspartates have been constructed for the GH6 enzymes Thermobifida fusca Cel6A (Tf Cel6A, at that time known as Thermomonospora fusca endocellulase E2) (20) and Cellulomonas fimi Cel6A (Cf Cel6A, known as CenA at the time) (21). Note that these studies were published in 1999 and 1995, respectively, before Koivula et al's seminal 2002 paper informing our current understanding of which residues perform the function of the catalytic base in this family of enzymes.…”

Characterization of Thermobifida fusca Cutinase-Carbohydrate-Binding Module Fusion Proteins and Their Potential Application in Bioscouring

“…The CBM of endoglucanase A (CenA) from C. fimi, which also belongs to the second family of CBMs, was shown to have high affinity to cellulose (12,15,39) and appears to be a suitable candidate. Subsequently, CBM CenA was fused to the C terminus of cutinase using the same linker from T. fusca Cel6A.…”

Surface Diffusion of Cellulases and Their Isolated Binding Domains on Cellulose

“…The gene fragments encoding the catalytically inactive mutant of the endoglucanase CenA (D252A) and the isolated CBD CenA were subcloned into the vector pUC18 and expressed in E. coli JM101 (10). Fermentations were carried out in a 20-liter Chemap fermenter at 37°C.…”

“…The endoglucanase CenA is moderately active on crystalline cellulose. A catalytically inactive mutant of CenA was therefore used to prevent surface degradation (10). This mutant binds substrate with wild-type affinity but is unable to cleave substrate because the acid catalyst Asp-252 is mutated to alanine.…”