We report the first X-ray structure of the unique “head-to-middle” monoterpene synthase, lavandulyl diphosphate synthase (LPPS). LPPS catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) to form lavandulyl diphosphate, a precursor to the fragrance lavandulol. The structure is similar to that of the bacterial cis-prenyl synthase, undecaprenyl diphosphate synthase (UPPS), and contains an allylic site (S1) in which DMAPP ionizes and a second site (S2) which houses the DMAPP nucleophile. Both S-thiolo-dimethylallyl diphosphate and S-thiolo-isopentenyl diphosphate bind intact to S2, but are cleaved to (thio)diphosphate, in S1. His78 (Asn in UPPS) is essential for catalysis and is proposed to facilitate diphosphate release in S1, while the P1 phosphate in S2 abstracts a proton from the lavandulyl carbocation to form the LPP product. The results are of interest since they provide the first structure and structure-based mechanism of this unusual prenyl synthase.
Angucyclines are
a structurally diverse class of actinobacterial
natural products defined by their varied polycyclic ring systems,
which display a wide range of biological activities. We recently discovered
lugdunomycin (
1
), a highly rearranged polyketide antibiotic
derived from the angucycline backbone that is synthesized via several
yet unexplained enzymatic reactions. Here, we show via
in
vivo
,
in vitro
, and structural analysis
that the promiscuous reductase LugOII catalyzes both a C6 and an unprecedented
C1 ketoreduction. This then sets the stage for the subsequent C-ring
cleavage that is key to the rearranged scaffolds of
1
. The 1.1 Å structures of LugOII in complex with either ligand
8-
O
-Methylrabelomycin (
4
) or 8-
O
-Methyltetrangomycin (
5
) and of apoenzyme
were resolved, which revealed a canonical Rossman fold and a remarkable
conformational change during substrate capture and release. Mutational
analysis uncovered key residues for substrate access, position, and
catalysis as well as specific determinants that control its dual functionality.
The insights obtained in this work hold promise for the discovery
and engineering of other promiscuous reductases that may be harnessed
for the generation of novel biocatalysts for chemoenzymatic applications.
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