Exploring the Structural and Dynamic Properties of a Chimeric Glycoside Hydrolase Protein in the Presence of Calcium Ions

GH10 xylanases and GH62 Arabinofuranosidases play key roles in the breakdown of arabinoxylans and are important tools in various industrial and biotechnological processes, such as renewable biofuel production, the paper industry, and the production of short-chain xylooligosaccharides (XOS) from plant biomass. However, the use of these enzymes in industrial settings is often limited due to their relatively low thermostability and reduced catalytic efficiency. To overcome these limitations, strategies based on enzymatic chimera construction and the use of metal ions and other cofactors have been proposed to produce new recombinant enzymes with improved catalytic activity and thermostability. Here, we examine the conformational dynamics of a GH10-GH62 chimera at different calcium ion concentrations through molecular dynamics simulations. While experimental data have demonstrated improved activity and thermostability in GH10-GH62 chimera, the mechanistic basis for these enhancements remains unclear. We explored the structural details of the binding subsites of Ca²⁺ in the parental enzymes GH62 from <i>Aspergillus fumigatus</i> (Afafu62) and a recombinant GH10 from <i>Cryptococcus flavescens</i> (Xyn10cf), as well as their chimeric combination, and how negatively charged electron pairing located at the protein surface affects Ca²⁺ capture. The results indicate that Ca²⁺ binding significantly contributes to structural stability and catalytic cavity modulation in the chimera, particularly evident at a concentration of 0.01 M. This effect, not observed in the parental GH10 and GH62 enzymes, highlights how Ca²⁺ enhances stability in the overall chimeric enzyme, while supporting a larger cavity volume in the chimera GH62 subunit. The increased catalytic site volume and reduced structural flexibility in response to Ca²⁺ suggest that calcium binding minimizes non-productive conformational states, which could potentially improve catalytic turnover. The findings presented here may aid in the development of more thermostable and efficient catalytic systems.