Identification and Mutation Analysis of Nonconserved Residues on the TIM-Barrel Surface of GH5_5 Cellulases for Catalytic Efficiency and Stability Improvement

Abstract

Exploring the potential functions of nonconserved residues on the outer side of α-helices and systematically optimizing them are pivotal for their application in protein engineering. Based on the evolutionary structural conservation analysis of GH5_5 cellulases, a practical molecular improvement strategy was developed. Highly variable sites on the outer side of the α-helices of the GH5_5 cellulase from Aspergillus niger (AnCel5A) were screened, and 14 out of the 34 highly variable sites were confirmed to exert a positive effect on the activity. After the modular combination of the positive mutations, the catalytic efficiency of the mutants was further improved. By using CMC-Na as the substrate, the catalytic efficiency and specific activity of variant AnCel5A_N193A/T300P/D307P were approximately 2.0-fold that of AnCel5A (227 ± 21 versus 451 ± 43 ml/s/mg and 1,726 ± 19 versus 3,472 ± 42 U/mg, respectively). The half-life (t_1/2) of variant AnCel5A_N193A/T300P/D307P at 75°C was 2.36 times that of AnCel5A. The role of these sites was successfully validated in other GH5_5 cellulases. Computational analyses revealed that the flexibility of the loop 6-loop 7-loop 8 region was responsible for the increased catalytic performance. This work not only illustrated the important role of rapidly evolving positions on the outer side of the α-helices of GH5_5 cellulases but also revealed new insights into engineering the proteins that nature left as clues for us to find. IMPORTANCE A comprehensive understanding of the residues on the α-helices of the GH5_5 cellulases is important for catalytic efficiency and stability improvement. The main objective of this study was to use the evolutionary conservation and plasticity of the TIM-barrel fold to probe the relationship between nonconserved residues on the outer side of the α-helices and the catalytic efficiency of GH5_5 cellulases by conducting structure-guided protein engineering. By using a four-step nonconserved residue screening strategy, the functional role of nonconserved residues on the outer side of the α-helices was effectively identified, and a variant with superior performance and capability was constructed. Hence, this study proved the effectiveness of this strategy in engineering GH5_5 cellulases and provided a potential competitor for industrial applications. Furthermore, this study sheds new light on engineering TIM-barrel proteins.

Keywords: GH5_5 family cellulases; TIM-barrel fold; catalytic efficiency; lignocellulosic biomass; nonconserved residues; α-helices.

FIG 1

Schematic representation of the strategy used in this study. Our strategy was comprised of four phases (I to IV). In phase I, cyan variable sites were generated with ConSurf algorithms, red conserved sites were eliminated, the sites on the outside the α-helices were selected for ΔΔG validation, and the unstable mutants in orange were filtered out. In phase II, the effective mutations were identified by experimental verification (mutations with cyan words). In phase III, the favorable mutations were grouped by helices, followed by a multipoint combination to filter out the unstable mutations. Then the most effective multipoint combination at α8 was selected by experimental validation. Finally, in the fourth phase, mutations between different helices were combined with α8 to achieve the target function (α8 in gray, and α5 in light green).

FIG 2

Visualization of identified amino acid positions in each phase. (A) In phase I, the 34 positions were identified at the outer side of the α-helices. (B) In phase II, 14 identified amino acid positions in AnCel5A. (C) In phase III, two beneficial positions (T300P and D307P in α8) were identified by grouped helices. (D) Three beneficial positions were confirmed from phase IV (N193A in α5, T300P and D307P in α8).

FIG 3

Correlation between computational and experimental screening. The difference in the proportion of amino acids before and after mutation at the 34 candidate sites, with the proportion of amino acids from the computational screening in red and the proportion of original amino acids in AnCel5A in black (left). The differences in ΔΔG prediction before and after mutation at the 34 candidate sites; if the values were >0, this indicates that the mutation is negative to enzyme stability, and if the values were <0, this indicates that the mutation is beneficial to enzyme stability (middle). The difference in specific activity between the 34 mutants and AnCel5A, with AnCel5A in black and the mutant in red (right).

FIG 4

The specific activities of mutants in phase III and phase IV. (A) The specific activities of the 34 participants in phase III. (B) In phase IV, the specific activities of the seven combinations.

FIG 5

Enzymatic properties of AnCel5A and the mutants. (A) Effects of pH on the enzyme activities evaluated at 75°C. (B) Effects of temperature on the enzyme activities evaluated at the indicated temperatures and pH 4.0.

FIG 6

Comparative RMSFs across all trajectories and the structure schematic. (A) RMSFs computed from MD simulations for AnCel5A and the T300P/D307P and N193A/T300P/D307P mutants at 300 K. (B) RMSFs computed from MD simulations for AnCel5A and the T300P/D307P and N193A/T300P/D307P mutants at 340 K. To highlight the differences in RMSFs between structural units, loop 6, α7-loop 7, and loop 8 regions are highlighted in blue, yellow, and green, respectively. (C) Top view of the structure schematic of loop 6, α7-loop 7, and loop 8 regions, which are highlighted in blue, yellow, and green, respectively. (D) Side view of the structure schematic of loop 6, α7-loop 7, and loop 8 regions. Mutant residues are shown as cyan sticks.

FIG 7

Conformational analysis of α7, α8, and loop 8 in the MD trajectory of WT and the mutants. (A) Specific interactions around the D307 and T300 residues of AnCel5A. (B) Structural effects of T300P/D307P on α7 and α8. (C) Comparison of residue conformations around W281 in AnCel5A and T300P/D307P. (D) Comparison of residue conformations around W287 in AnCel5A and T300P/D307P.

FIG 8

Time-course hydrolysis of PASC. Time profiling of reducing sugars production was detected every 0.5 h for a total of 2 h under pH 5.0 and 50°C.

FIG 9

Schematic diagram of AnCel5A. (A) Top view of AnCel5A. The catalytic residues Glu142 and Glu248 are shown as sticks. The residues are colored according to the conservation scores as shown in the Color code. The most variable residues are colored in turquoise, and the frequency of which is approximately 0–30%, while the most conserved residues are colored in maroon, and the frequency of which is approximately 60–90%. (B) Side view of AnCel5A; the residues are colored according to the conservation scores. (C) Top view of AnCel5A; the residues are colored according to the conservation scores. (D) Side view of AnCel5A. The residues on the outer side of the α-helices are colored cyan.