Polyketides are a class of
natural products with diverse structures and biological activities. The structural variability of
aromatic products of
fungal nonreducing, multidomain iterative
polyketide synthases (NR-PKS group of IPKSs) results from regiospecific
cyclizations of reactive poly-beta-keto intermediates. How poly-beta-keto species are synthesized and stabilized, how their chain lengths are determined, and, in particular, how specific
cyclization patterns are controlled have been largely inaccessible and functionally unknown until recently. A product template (PT) domain is responsible for controlling specific
aldol cyclization and
aromatization of these mature
polyketide precursors, but the mechanistic basis is unknown. Here we present the 1.8 A
crystal structure and mutational studies of a
dissected PT monodomain from PksA, the NR-PKS that initiates the
biosynthesis of the potent hepatocarcinogen
aflatoxin B(1) in
Aspergillus parasiticus. Despite having minimal
sequence similarity to known
enzymes, the structure displays a distinct 'double hot dog' (DHD) fold. Co-crystal structures with
palmitate or a
bicyclic substrate mimic illustrate that PT can bind both linear and
bicyclic polyketides. Docking and mutagenesis studies reveal residues important for
substrate binding and
catalysis, and identify a
phosphopantetheine localization channel and a deep two-part interior binding pocket and reaction chamber.
Sequence similarity and extensive conservation of
active site residues in PT domains suggest that the mechanistic insights gleaned from these studies will prove general for this class of IPKSs, and lay a foundation for defining the molecular rules controlling NR-PKS
cyclization specificity.
