Computational engineering of cellulase Cel9A-68 functional motions through mutations in its linker region

Abstract

Microbial cellulosic degradation by cellulases has become a complementary approach for biofuel production. However, its efficiency is hindered by the recalcitrance of cellulose fibres. In this context, computational protein design methods may offer an efficient way to obtain variants with improved enzymatic activity. Cel9A-68 is a cellulase from Thermobifida fusca that is still active at high temperatures. In a previous work, we described a collective bending motion, which governs the overall cellulase dynamics. This movement promotes the approximation of its CBM and CD structural domains (that are connected by a flexible linker). We have identified two residues (G460 and P461) located at the linker that act as a hinge point. Herein, we applied a new level of protein design, focusing on the modulation of this collective motion to obtain cellulase variants with enhanced functional dynamics. We probed whether specific linker mutations would affect Cel9A-68 dynamics through computational simulations. We assumed that P461G and G460+ (with an extra glycine) constructs would present enhanced interdomain motions, while the G460P mutant would be rigid. From our results, the P461G mutation resulted in a broader exploration of the conformational space, as confirmed by clustering and free energy analyses. The WT enzyme was the most rigid system. However, G460P and P460+ explored distinct conformational states described by opposite directions of low-frequency normal modes; they sampled preferentially closed and open conformations, respectively. Overall, we highlight two significant findings: (i) all mutants explored larger conformational spaces than the WT; (ii) the selection of distinct conformational populations was intimately associated with the mutation considered. Thus, the engineering of Cel9A-68 motions through linker mutations may constitute an efficient way to improve cellulase activity, facilitating the disruption of cellulose fibres.

Bioengineered Enzymes and Precision Fermentation in the Food Industry

“…Computer-aided enzyme simulation enabled to redesign of Cel9A-68 cellulase from Thermobifida fusca with improved cellulase activity. Such features could be extremely relevant for cellulose degradation in the brewery sector [ 26 ]. A lipase from P. aeruginosa PAO1 was recently computationally “reverse-engineered” using proline mutations.…”

Progress in Ameliorating Beneficial Characteristics of Microbial Cellulases by Genetic Engineering Approaches for Cellulose Saccharification

“…The presence of more flexible linkers resulted in higher cellulolytic activity. Thus, the linker mutations may prove to be an efficient way to improve enzyme activity (Costa et al, 2018).…”

An overview of biomass conversion: exploring new opportunities

“…Based on the previous study, Costa, Silva & Batista (2018) identified two residues (Gly460 and Pro461) located at the linker that act as a hinge point and assumed that a Pro461Gly and a Gly460+ (with an extra glycine) constructs of a Cel9A-68 would present enhanced interdomain motions, while the Gly460Pro mutant would be rigid. The Pro461Gly mutation resulted in an extension of the conformational space, as confirmed by clustering and free energy analyses.…”