A Radical Clock Probe Uncouples H-atom Abstraction from Thi-oether Crosslink Formation by the Radical SAM Enzyme SkfB

Will Kincannon, Nathan A. Bruender, Vahe Bandarian

Research output: Contribution to journalArticle

Abstract

Sporulation killing factor (SKF) is a ribosomally synthesized and post-translationally modified pep-tide (RiPP) produced by Bacillus. SKF contains a thioether crosslink between the α-carbon at position 40 and thiol of Cys32, introduced by a member of the radical S-adenosyl-L-methionine (SAM) super-family, SkfB. Radical SAM enzymes employ a 4Fe-4S cluster to bind and reductively cleave SAM to generate a 5'-deoxyadenosyl radical. SkfB lever-ages this radical intermediate to abstract the α-H-atom at Met40 to initiate crosslinking. In addition to the cluster that binds SAM, SkfB also has an auxil-iary cluster, the function of which is not known. We demonstrate that a substrate analog with a cy-clopropylglycine (CPG) moiety replacing the wildtype Met40 sidechain forgoes thioether cross-linking for an alternative radical ring opening of the CPG sidechain. The ring opening reaction also takes place with a catalytically inactive SkfB variant in which the auxiliary Fe-S cluster is absent. There-fore, CPG containing peptide uncouples H-atom abstraction from thioether bond formation, limiting the role of the auxiliary cluster to promoting thi-oether crosslink formation. CPG proves a valuable tool to uncouple H-atom abstraction from peptide modification in RiPP maturases, as well as demon-strates potential to leverage RS enzyme reactivity to create noncanonical amino acids.

Original languageEnglish (US)
JournalBiochemistry
DOIs
StateAccepted/In press - May 10 2018

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S-Adenosylmethionine
Clocks
Sulfides
Atoms
Enzymes
Peptides
Tides
Bacilli
Sulfhydryl Compounds
Crosslinking
Bacillus
Carbon
Amino Acids
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

A Radical Clock Probe Uncouples H-atom Abstraction from Thi-oether Crosslink Formation by the Radical SAM Enzyme SkfB. / Kincannon, Will; Bruender, Nathan A.; Bandarian, Vahe.

In: Biochemistry, 10.05.2018.

Research output: Contribution to journalArticle

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