Beyond the Active Site: The addition of a remote loop reveals a new complex biological function for chitinase enzymes
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Abstract
Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, our understanding of loop functions is mainly limited to the catalytic loops. To understand the function of remote loops in enzymes, we studied Glycoside hydrolase family 19 (GH19) chitinase - an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discovered that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrated that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that Nature applies this new strategy at the molecular level to achieve a complex biological function while maintaining the original activity one in the catalytic pocket, suggesting an alternative way to design new enzyme function.
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Thank you so much for finding our study interesting. We have uploaded the revised manuscript.
*It's making me wonder whether this could either be because it recognizes the substrate in a particular structural conformation unique to the in vivo cell wall. *
We believe that accessibility to the cell wall is critical to perform antifungal activity. We are not sure if proteins recognize the specific cell wall conformation or if the cell wall of the fungus we used in this study is easily accessible or not, as GH chitinases do not show antifungal activity on some fungi. It might be due to the complexity and the composition of the cell walls, which vary from species to species.
Or perhaps this long-range interaction allows a different processive activity along a cell wall that is required for lytic/anti-fungal activity?
GH19 chitinases are not …
Thank you so much for finding our study interesting. We have uploaded the revised manuscript.
*It's making me wonder whether this could either be because it recognizes the substrate in a particular structural conformation unique to the in vivo cell wall. *
We believe that accessibility to the cell wall is critical to perform antifungal activity. We are not sure if proteins recognize the specific cell wall conformation or if the cell wall of the fungus we used in this study is easily accessible or not, as GH chitinases do not show antifungal activity on some fungi. It might be due to the complexity and the composition of the cell walls, which vary from species to species.
Or perhaps this long-range interaction allows a different processive activity along a cell wall that is required for lytic/anti-fungal activity?
GH19 chitinases are not processive enzymes. Here is a summary of how GH19 chitinases acquire antifungal activity:
We have identified six residues in close proximity to loop II that establish new interactions with this loop (new Figure 4C). To elucidate the functional implications of these interactions, we constructed mutants by introducing substitutions for these six residues into Anc4 + Loop II.
Our comprehensive analysis, encompassing antifungal activity assays and binding activity experiments on these mutants, demonstrated a significant trend. As illustrated in Table 2, we observed a gradual and noteworthy increase in antifungal activity with the stepwise introduction of these substitutions. Integrating all six residues increased binding activity to the fungal cell wall (Figure 4E). We used cell wall fractions (cell wall fragments) in this binding assay.
Furthermore, our molecular dynamics (MD) simulations provided additional insights as depicted in Figure 4F. These simulations showcased a gradual reduction in the flexibility of loop II regions as a direct consequence of these substitutions.
In summary, GH19 chitinase acquires antifungal properties through the interplay of distal loop insertion and the strategic substitutions that stabilize the loop conformation, ultimately optimizing it for enhanced antifungal activity.
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In the evolution of GH19 chitinase, stabilizing the key remote loop regions is important to perform new protein functions
Would be interested to see whether our protein structural comparison tool Protein Cartography could help you visualize a structural/evolutionary landscape of this family, and possibly use your insights to identify more GH19 chitinases that are potential antifungal enzymes!
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lose chitin degrading activity in the fungal cell wall
This is super interesting. It's making me wonder whether this could either be because it recognizes the substrate in a particular structural conformation unique to the in vivo cell wall. Or perhaps this long-range interaction allows a different processive activity along a cell wall that is required for lytic/anti-fungal activity? Did you ever compare binding of this mutant vs control to cell wall fragments, as opposed to intact cells? Would be interesting to see whether this phenotype is dependent on the state of the substrate.
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