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.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Zhu W,Martins AM,Klinman JPdoi
10.1016/bs.mie.2018.04.002subject
Has Abstractpub_date
2018-01-01 00:00:00pages
389-420eissn
0076-6879issn
1557-7988pii
S0076-6879(18)30136-8journal_volume
606pub_type
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