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Abstract
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Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) has an Ig-like domain and the enzyme stability
is dependent to calcium. In this study the effect of calcium on the structure and stability of the wild-type enzyme and the
truncated form (the wild-type enzyme without Ig-like domain, AaCel9AΔN) was investigated. Fluorescence quenching
results indicated that calcium increased and decreased the rigidity of the wild-type and truncated enzymes, respectively.
RMSF results indicated that AaCel9A has two flexible regions (regions A and B) and deleting the Ig-like domain increased
the truncated enzyme stability by decreasing the flexibility of region B probably through increasing the hydrogen bonds.
Calcium contact map analysis showed that deleting the Ig-like domain decreased the calcium contacting residues and their
calcium binding affinities, especially, in region B which has a role in calcium binding site in AaCel9A. Metal depletion and
activity recovering as well as stability results showed that the structure and stability of the wild-type and truncated enzymes
are completely dependent on and independent of calcium, respectively. Finally, one can conclude that the deletion of Iglike
domain makes AaCel9AΔN independent of calcium via decreasing the flexibility of region B through increasing the
hydrogen bonds. This suggests a new role for the Ig-like domain which makes AaCel9A structure dependent on calcium.
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