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A Radical Clock Probe Uncouples H Atom Abstraction from Thioether Cross-Link Formation by the Radical S‑Adenosyl‑l‑methionine Enzyme SkfB
journal contribution
posted on 2018-07-02, 00:00 authored by William M. Kincannon, Nathan A. Bruender, Vahe BandarianSporulation
killing factor (SKF) is a ribosomally synthesized and
post-translationally modified peptide (RiPP) produced by Bacillus. SKF contains a thioether cross-link between the α-carbon
at position 40 and the thiol of Cys32, introduced by a member of the
radical S-adenosyl-l-methionine (SAM) superfamily,
SkfB. Radical SAM enzymes employ a 4Fe–4S cluster to bind and
reductively cleave SAM to generate a 5′-deoxyadenosyl radical.
SkfB utilizes this radical intermediate to abstract the α-H
atom at Met40 to initiate cross-linking. In addition to the cluster
that binds SAM, SkfB also has an auxiliary cluster, the function of
which is not known. We demonstrate that a substrate analogue with
a cyclopropylglycine (CPG) moiety replacing the wild-type Met40 side
chain forgoes thioether cross-linking for an alternative radical ring
opening of the CPG side chain. The ring opening reaction also takes
place with a catalytically inactive SkfB variant in which the auxiliary
Fe–S cluster is absent. Therefore, the CPG-containing peptide
uncouples H atom abstraction from thioether bond formation, limiting
the role of the auxiliary cluster to promoting thioether cross-link
formation. CPG proves to be a valuable tool for uncoupling H atom
abstraction from peptide modification in RiPP maturases and demonstrates
potential to leverage RS enzyme reactivity to create noncanonical
amino acids.
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Keywords
peptide modificationα- H atomsubstrate analogueCPG side chainMet 40SKFring opening reactionα- carbonThioether Cross-Link Formationthioether cross-linkring openinguncoupling H atom abstractionCPG-containing peptide uncouples H atom abstractionFethioether cross-link formationclusterSkfB variantthioether bond formationleverage RS enzyme reactivityRadical SAM enzymesRadical Clock Probe Uncouples H Atom AbstractionRiPP maturasesposition 40wild-type Met 40 side chainthioether cross-linking
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