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Functional analysis of the C-terminal positive region in an archaeal GH46 family chitosanase
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DOI：10.1016/j.ijbiomac.2026.152463.png)
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Abstract
En 中文
Archaeal enzymes often possess unique structural features. Here, we identified an archaeal chitosanase from Methanosarcina thermophila, mtCsn46, which contains a charged C-terminal extension. Experimental results showed that the positively charged region (PR) plays a critical role in enzyme function. Molecular dynamics (MD) simulations revealed that PR is positioned above the substrate-binding pocket, facilitating substrate hydrolysis and enhancing interactions that increase the degree of polymerization of chitooligosaccharides. A rationally designed Q19E mutation strengthened interactions between PR and the enzyme core, improving structural stability and elevating the optimal operating temperature. Although substrate affinity decreased, enzymatic activity increased nearly 2-fold, with a Vmax of 4129.00 μmol L−1 min−1. MD analysis indicated that Glu19 forms stable salt bridges with Lys244 and Lys245, accounting for the enhanced stability. These findings clarify the functional significance of the unique C-terminal extension in archaeal chitosanases and provide a structure-based strategy for enzyme engineering.
Keywords:
archaeal chitosanase
C-terminal extension
molecular dynamics simulations
enzyme engineering
substrate hydrolysis
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