Structural Analysis of a Non-canonical Terpene Synthase.

Natural products generated by biosynthetic enzymes provide complex structural diversity. Several fungal species have evolved to obtain this diversity by combining several core enzymes in a biosynthetic pathway, including polyketide synthases (PKS), nonribosonal peptides synthases (NRPS), and terpene cyclases (TC) along with accessorizing enzymes such as transferases and oxidoreductases. Combinations of different core enzymes in hybrid gene clusters affords structurally complex scaffolds unachievable with a single core enzyme. Recently, the Tang laboratory characterized a nine-gene cluster from the fungal species, Aspergillus flavus, which encodes for two TCs, FlvE and FlvF, along with a single-module NRPS, FlvI. Additionally, the flvfcluster contains multiple redox enzymes, including the short-chain reductase FlvB, two P450s (FlvC and FlvD), and a didomain enzyme (FlvA) comprising a PLP-dependent lyase and a non-heme Fe α-ketoglutarate-dependent oxygenase. Other enzymes in the cluster include FlvG, a homolog of ornithine decarboxylase, and FlvH, a lysine N-methyltransferase. Together, the flvf pathway produces flavunoidine, an alkaloidal terpenoid containing a tetracyclic and oxygenated sesquiterpene core connected to dimethylcadaverine and acylated with 5,5-dimethyl-L-pipecolate. Notably, a unique feature of the flvf pathway is the enzyme, FlvF. Although annotated as a terpene cyclase, FlvF catalyzes the stereospecific C-N bond formation attaching dimethylcadaverine to the sesquiterpene core, an unusual reaction given that FlvF has been proposed to take a secondary carbocation as substrate. The mechanism underlying this reaction has not been reported in any other terpene cyclase and is not well understood. Herein, we report the first crystal structure of FlvF determined at 2.6 Å resolution. The initial electron density map was phased using a search model generated by AlphaFold2 (with access provided by ColabFold) and refined with an R /R of 0.22/0.27, respectively. The structure adopts an alpha-fold of class I terpene cyclases, and a search for structural homologs using the DALI server identifies the closest matches as the terpene cyclases FgGS, PaFS, and aristolochene synthase. Notably, FlvF does not contain the canonical metal-binding motifs observed in class I cyclases. A bound molecule of Tris forms cation-pi interactions with surrounding aromatic residues at the base of the active site, which might similarly stabilize a carbocation intermediate. Further analysis of the active site indicates multiple possibilities for general acid catalysis. Future structural and mechanistic studies are necessary to elucidate the mechanism of this enzyme.