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Methods for Expression, Purification, and Characterization of PqqE, a Radical SAM Enzyme in the PQQ Biosynthetic Pathway.

  • Author(s): Zhu, Wen
  • Martins, Ana M
  • Klinman, Judith P
  • et al.

Published Web Location

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287933/
No data is associated with this publication.
Abstract

PqqE is the first enzyme in the biosynthetic pathway of the redox cofactor pyrroloquinoline quinone (PQQ), catalyzing the formation of a carbon-carbon bond in the precursor peptide PqqA. PqqE is a radical S-adenosyl-l-methionine (SAM) (RS) enzyme, a family of enzymes that use the reductive cleavage of a [4Fe-4S] cluster-bound SAM molecule to generate a 5'-deoxyadenosyl radical. This radical is then used to initiate an array of reactions that otherwise would be unlikely to occur. PqqE is a founding member of a subset family of RS enzymes that, additionally to the SAM [4Fe-4S] cluster, have a SPASM domain containing additional, auxiliary Fe-S clusters. Most radical SAM enzymes are highly sensitive to oxygen, which destroys their Fe-S clusters. This can pose several limitations when working with these enzymes, since most of the work has to be done under anaerobic conditions. Here, we summarize the methods developed in our lab for the expression and purification of PqqE. We also highlight the several methods we have used for the characterization of the enzyme.

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