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Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.

Md Anarul Hoque1, Yong Zhang1, Liuqing Chen1

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Researchers engineered novel biocatalysts by inserting residues into enzyme active sites. This stepwise loop insertion strategy (StLois) significantly enhanced enzyme efficiency and altered substrate specificity for organophosphate hydrolysis.

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Area of Science:

  • Enzyme engineering
  • Biocatalysis
  • Protein engineering

Background:

  • Remodeling enzyme active sites to create novel biocatalysts is a significant challenge.
  • Active site loops play a crucial role in determining enzyme function and specificity.

Purpose of the Study:

  • To develop and evaluate a stepwise loop insertion strategy (StLois) for enzyme functional remodeling.
  • To investigate the potential of StLois in altering the catalytic efficiency and substrate specificity of phosphotriesterase-like lactonase (GkaP-PLL).

Main Methods:

  • Developed the stepwise loop insertion strategy (StLois) involving randomized residue pair insertion into active site loops.
  • Applied StLois to the Geobacillus kaustophilus phosphotriesterase-like lactonase (GkaP-PLL).
  • Generated and characterized enzyme variants, including ML7-B6, through targeted loop modifications.

Main Results:

  • The variant ML7-B6, with six residues inserted into active site loop 7, showed a 16-fold increase in catalytic efficiency towards ethyl-paraoxon.
  • Achieved a >10^7 fold substrate specificity shift relative to the wild-type lactonase.
  • Remodeled variants exhibited a 760-fold increase in activity against organophosphates like parathion, diazinon, and chlorpyrifos.
  • Structural and docking studies elucidated the mechanism behind the inverted enzyme specificity.

Conclusions:

  • The stepwise loop insertion strategy (StLois) is effective for rapidly generating novel enzyme functions.
  • Loop remodeling offers a powerful approach for tailoring enzyme specificity and enhancing catalytic activity.
  • This strategy holds significant potential for the development of new biocatalysts for various applications.