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Development of an

Mary C Andorfer1,2, Devin T King-Roberts3, Christa N Imrich4

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Summary
This summary is machine-generated.

Researchers identified a soluble enzyme, 4-isopropylbenzylsuccinate synthase-activating enzyme (IbsAE), enabling in vitro study of hydrocarbon-degrading enzymes (XSSs). This breakthrough allows biochemical exploration and engineering of XSSs for biocatalysis.

Keywords:
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Area of Science:

  • Biochemistry
  • Enzymology
  • Microbial Metabolism

Background:

  • Anaerobic hydrocarbon degradation is initiated by X-succinate synthases (XSSs) that add hydrocarbons to fumarate.
  • XSSs require a glycyl radical cofactor installed by an activating enzyme (XSS-AE).
  • Previous in vitro studies were limited by the insolubility of XSS-AEs.

Purpose of the Study:

  • To identify a soluble XSS-AE for in vitro biochemical studies.
  • To enable biochemical characterization and engineering of XSSs.
  • To investigate the role of enzyme subunits in XSS activity.

Main Methods:

  • Genome mining to identify a novel XSS-AE.
  • Soluble expression of the identified XSS-AE in Escherichia coli.
  • In vitro biochemical assays using purified enzymes.

Main Results:

  • Identified and expressed a soluble 4-isopropylbenzylsuccinate synthase-AE (IbsAE).
  • Demonstrated that soluble IbsAE can activate both IBSS and benzylsuccinate synthase (BSS) in vitro.
  • Found that the beta subunit of BSS accelerates hydrocarbon addition.

Conclusions:

  • The development of soluble XSS-AEs overcomes a major hurdle in studying XSS enzymes.
  • This work provides a platform for the biochemical exploration of XSSs.
  • The methodology can be applied to engineer XSSs for synthetic biocatalysis.