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Defining Substrate Specificities for Lipase and Phospholipase Candidates
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Structural Insight into Acyl-ACP Thioesterase toward Substrate Specificity Design.

Yanbin Feng1, Yayue Wang1,2, Jiao Liu1,2

  • 1Marine Bioengineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.

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|October 10, 2017
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Summary

Scientists elucidated the substrate selection mechanism of acyl-ACP thioesterase (TE) by determining the crystal structure of Umbellularia californica FatB. Redesigning this enzyme enhanced C14 fatty acid production in engineered E. coli.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Acyl-ACP thioesterases (TEs) are crucial enzymes in type II fatty acid synthesis, controlling fatty acid chain length by hydrolyzing thioester bonds.
  • While most TEs recognize C16:0 and C18:1 substrates, specific TEs modulate elongation at C8-C14, but their acyl selection mechanism remains unclear.

Purpose of the Study:

  • To elucidate the substrate selection mechanism of acyl-ACP thioesterase (TE).
  • To determine the crystal structure of the C12-specific thioesterase FatB from Umbellularia californica (UcFatB).
  • To rationally redesign UcFatB for altered fatty acid chain length selectivity.

Main Methods:

  • X-ray crystallography was used to determine the structure of UcFatB.
  • Site-directed mutagenesis was employed to alter amino acid residues within the substrate binding pocket.
  • Enzyme activity assays and engineered E. coli strains were utilized to assess substrate selectivity and fatty acid production.

Main Results:

  • The crystal structure revealed two hotdog domains, with an Asp-His-Glu catalytic network in the C-terminal domain and the substrate binding pocket in the N-terminal domain.
  • Key unconservative amino acids on β5, β2, β4 sheets and T137 on the α1 helix were identified as critical for UcFatB's C12 specificity.
  • The T137G mutant showed increased activity on C14 substrates in vitro and resulted in up to 40% C14 fatty acid content in engineered E. coli, compared to 10% for the wild type.

Conclusions:

  • The study unraveled the substrate selection mechanism of UcFatB, highlighting the role of specific amino acids in accommodating different fatty acid chain lengths.
  • Rational redesign of the substrate binding pocket by mutating T137G successfully shifted the enzyme's selectivity towards C14 fatty acids.
  • This work provides a novel strategy for engineering fatty acid synthesis pathways to tailor fatty acid production for various applications.