Effect of N-terminal modification on the mode of action between the Xyn11A and Xylotetraose
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Author list: Ngenyoung A., Muhammad A., Rattanarojpong T., Sutthibutpong T., Khunrae P.
Publisher: Elsevier
Publication year: 2021
Journal: International Journal of Biological Macromolecules (0141-8130)
Volume number: 170
Start page: 240
End page: 247
Number of pages: 8
ISSN: 0141-8130
Languages: English-Great Britain (EN-GB)
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Abstract
The purpose of this study was to gain an insight into the effects of mutation-induced binding pocket tilting of the Xyn11A xylanase from Bacillus firmus K-1 in producing a unique hydrolysis characteristic. In this study, the wildtype Xyn11A and its K40L mutant were compared for their hydrolysis patterns on beechwood xylan and xylooligosaccharides of sizes 2 to 6. According to our thin-layer chromatography experiment, the K40L mutant produced a larger amount of xylotetraose leftover than the wildtype. Kinetic determination of the WT and K40L mutant suggested that the higher X4 leftover on TLC was reflected in the decreasing catalytic efficiency (kcat/Km) between enzyme and X4. The mechanisms underlying this efficiency loss were examined through atomistic molecular dynamics (MD) simulations. The MD trajectory analysis showed that the mutation-induced binding pocket tilting resulted in an additional hydrophobic contact between the reducing end of X4 and Trp128. Meanwhile, the interactions between the non-reducing end and the Arg112 residue near the active site became lost, which could decrease the catalytic efficiency. This work suggested that the protein engineering to fine-tune the hydrolysis pattern for some desired xylooligosaccharide products was possible. © 2020 Elsevier B.V.
Keywords
Xylooligosaccharides
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