Cel48A from Thermobifida fusca: Structure and site directed mutagenesis of key residues

Abstract

Lignocellulosic biomass is a potential source of sustainable transportation fuels, but efficient enzymatic saccharification of cellulose is a key challenge in its utilization. Cellulases from the glycoside hydrolase (GH) family 48 constitute an important component of bacterial biomass degrading systems and structures of three enzymes from this family have been previously published. We report a new crystal structure of TfCel48A, a reducing end directed exocellulase from Thermobifida fusca, which shows that this enzyme shares important structural features with the other members of the GH48 family. The active site tunnel entrance of the known GH48 exocellulases is enriched in aromatic residues, which are known to interact well with anhydroglucose units of cellulose. We carried out site-directed mutagenesis studies of these aromatic residues (Y97, F195, Y213, and W313) along with two non-aromatic residues (N212 and S311) also located around the tunnel entrance and a W315 residue inside the active site tunnel. Only the aromatic residues located around the tunnel entrance appear to be important for the ability of TfCel48A to access individual cellulose chains on bacterial cellulose (BC), a crystalline substrate. Both Trp residues were found to be important for the processivity of TfCel48A on BC and phosphoric acid swollen cellulose (PASC), but only W313 appears to play a role in the ability of the enzyme to access individual cellulose chains in BC. When acting on BC, reduced processivity was found to correlate with reduced enzyme activity. The reverse, however, is true when PASC is the substrate. Presumably, higher density of available cellulose chain ends and the amorphous nature of PASC explain the increased initial activity of mutants with lower processivity.

Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity

“…The processivity of the wild type TrCel7A on Avicel has been measured before (15, 22, 31), and the current result is in line with these earlier reports. We note that relationships between product profiles and processivity have been extensively discussed (4,35,37), and somewhat deviant principles for the calculation of n have been put forward (22,31,36,38). A discussion of the validity and weaknesses of these strategies is beyond the current scope, but using different approaches, we consistently found that the processivity of W38A was about 25% lower than the wild type.…”

“…3 showed a crossover at intermediate substrate loads, it appears that a broader range of loads may be necessary to reach a conclusion on the effect of the mutation on activity. We emphasize, however, that the W38A mutants studied here for their activity against Avicel (with an intermediate crystallinity) could behave differently on other substrates and hence that further experimental work would be required to elucidate possible kinetic differences between Trp mutations near the opening (16,38,44) and (as in the middle of the catalytic tunnel, respectively.…”

“…Many mutational studies in which Trp has been replaced with nonaromatic residues have shown negative effects on activity and processivity of, for example, cellulases (16,36,38,(43)(44)(45)(46) and chitinases (47)(48)(49). One suggested role of Trp is that "stacking" interactions of -electrons in its aromatic ring and the ␣-face of the carbohydrate (50) enable the necessary sliding of the bound chain (4, 51), possibly through stabilization of the transition state between adjacent positions in the processive movement (52,53).…”

“…One suggested role of Trp is that "stacking" interactions of -electrons in its aromatic ring and the ␣-face of the carbohydrate (50) enable the necessary sliding of the bound chain (4, 51), possibly through stabilization of the transition state between adjacent positions in the processive movement (52,53). Tryptophan residues near the entrance of the active tunnel are also important for the initial "threading" and recognition of the cellulose strand, particularly on crystalline substrates (16,26,38,44,54). In the current work, we used the W38A mutation in T. reesei Cel7A to obtain an enzyme with weakened substrate affinity.…”

“…Some studies on processive cellulases have found that replacement of Trp residues near the entrance of the catalytic tunnel leads to enhanced activity against soluble and/or amorphous substrates, whereas the hydrolysis of highly crystalline substrate is reduced in comparison with the wild type (16,38,44). In these cases, the activity was only tested at one (low) substrate load (0.7-2.5 g/liter).…”