Among
the ribosomally synthesized and post-translationally modified
peptide (RiPP) natural products, “graspetides” (formerly
known as microviridins) contain macrocyclic esters and amides that
are formed by ATP-grasp ligase tailoring enzymes using the side chains
of Asp/Glu as acceptors and Thr/Ser/Lys as donors. Graspetides exhibit
diverse patterns of macrocylization and connectivities exemplified
by microviridins, that have a caged tricyclic core, and thuringin
and plesiocin that feature a “hairpin topology” with
cross-strand ω-ester bonds. Here, we characterize chryseoviridin,
a new type of multicore RiPP encoded by Chryseobacterium
gregarium DS19109 (Phylum Bacteroidetes) and solve
a 2.44 Å resolution crystal structure of a quaternary complex
consisting of the ATP-grasp ligase CdnC bound to ADP, a conserved
leader peptide and a peptide substrate. HRMS/MS analyses show that
chryseoviridin contains four consecutive five- or six-residue macrocycles
ending with a microviridin-like core. The crystal structure captures
respective subunits of the CdnC homodimer in the apo or substrate-bound
state revealing a large conformational change in the B-domain upon
substrate binding. A docked model of ATP places the γ-phosphate
group within 2.8 Å of the Asp acceptor residue. The orientation
of the bound substrate is consistent with a model in which macrocyclization
occurs in the N- to C-terminal direction for core peptides containing
multiple Thr/Ser-to-Asp macrocycles. Using systematically varied sequences,
we validate this model and identify two- or three-amino acid templating
elements that flank the macrolactone and are required for enzyme activity
in vitro. This work reveals the structural basis for ω-ester
bond formation in RiPP biosynthesis.
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