Loops around the Heme Pocket Have a Critical Role in the Function and Stability of BsDyP from Bacillus subtilis.

Carolina F Rodrigues,Magali F Scocozza,Diogo Silva,Carlos Frazão,Vânia Brissos,Patrícia T Borges,Lígia O Martins,André Taborda

doi:10.3390/ijms221910862

Abstract

Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme’s overall stability by 2 kcal mol−1, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.

Highlights

Biocatalysis is both a green and sustainable technology and redox biocatalysts offer eco-friendly advantages in comparison with conventional chemical reactions due to the selectivity, controllability and economy of their reactions
Sci. 2021, 22, 10862 probably corresponds to the unprocessed cytoplasmic precursor containing the signal peptide, and a lower band that corresponds to the mature periplasmic enzyme without the signal peptide [29], to what was observed in the heterologous expression of TfuDyP Thermobifida fusca [31] and E. coli YcdB [32], DyP members from class I harboring an N-terminal twin-arginine sequence
The results indicate that the variation in the loops’ flexibility and the widening of the active site entrance in the evolved variant 5G5 negatively affect substrate binding, as reflected in higher Michaelis constants, Km, but simultaneously facilitate electron transfer from substrates to the heme, as assessed by the increased catalytic rates of 5G5 for DMP

Summary

Introduction

Biocatalysis is both a green and sustainable technology and redox biocatalysts offer eco-friendly advantages in comparison with conventional chemical reactions due to the selectivity, controllability and economy of their reactions. DyPs are a family of microbial heme peroxidases that display structural features analogous to chlorite dismutases with an α + β ferredoxin-like fold [12]. These enzymes show a remarkably broad range of substrates, from synthetic azo and antraquinonic dyes and aromatic sulfides to iron and manganese ions, phenolic and nonphenolic lignin units, wheat straw lignocellulose and kraft lignin, and are interesting enzymes for a vast array of biotechnological applications including bioprocesses targeted at the valorization of lignin [9,13,14,15]. DyPs are classified into four distinct classes based on their primary structure, with A–C subfamilies of bacterial origin and the D subfamily of fungal origin [16]

Methods

Results

Conclusion