Sactipeptides are ribosomally synthesized
peptides that contain
a characteristic thioether bridge (sactionine bond) that is installed
posttranslationally and is absolutely required for their antibiotic
activity. Sactipeptide biosynthesis requires a unique family of radical
SAM enzymes, which contain multiple [4Fe-4S] clusters, to form the
requisite thioether bridge between a cysteine and the α-carbon
of an opposing amino acid through radical-based chemistry. Here we
present the structure of the sactionine bond-forming enzyme CteB,
from <i>Clostridium thermocellum ATCC 27405</i>, with both
SAM and an N-terminal fragment of its peptidyl-substrate at 2.04 Å
resolution. CteB has the (β/α)<sub>6</sub>-TIM barrel
fold that is characteristic of radical SAM enzymes, as well as a C-terminal
SPASM domain that contains two auxiliary [4Fe-4S] clusters. Importantly,
one [4Fe-4S] cluster in the SPASM domain exhibits an open coordination
site in absence of peptide substrate, which is coordinated by a peptidyl-cysteine
residue in the bound state. The crystal structure of CteB also reveals
an accessory N-terminal domain that has high structural similarity
to a recently discovered motif present in several enzymes that act
on ribosomally synthesized and post-translationally modified peptides
(RiPPs), known as a RiPP precursor peptide recognition element (RRE).
This crystal structure is the first of a sactionine bond forming enzyme
and sheds light on structures and mechanisms of other members of this
class such as AlbA or ThnB