Aromatic polyketides make up a large class of natural products with diverse bioactivity. During biosynthesis, linear poly-β-ketone intermediates are regiospecifically cyclized, yielding molecules with defined cyclization patterns that are crucial for polyketide bioactivity. The aromatase/cyclases (ARO/CYCs) are responsible for regiospecific cyclization of bacterial polyketides. The two most common cyclization patterns are C7-C12 and C9-C14 cyclizations. We have previously characterized three monodomain ARO/CYCs: ZhuI, TcmN, and WhiE. The last remaining uncharacterized class of ARO/CYCs is the di-domain ARO/CYCs, which catalyze C7-C12 cyclization and/or aromatization. Di-domain ARO/CYCs can further be separated into two subclasses: "nonreducing" ARO/CYCs, which act on nonreduced poly-β-ketones, and "reducing" ARO/CYCs, which act on cyclized C9 reduced poly-β-ketones. For years, the functional role of each domain in cyclization and aromatization for di-domain ARO/CYCs has remained a mystery. Here we present what is to our knowledge the first structural and functional analysis, along with an in-depth comparison, of the nonreducing (StfQ) and reducing (BexL) di-domain ARO/CYCs. This work completes the structural and functional characterization of mono-and didomain ARO/CYCs in bacterial type II polyketide synthases and lays the groundwork for engineered biosynthesis of new bioactive polyketides.polyketide biosynthesis | structural biology | aromatase/cyclase T he biosynthesis of type II aromatic polyketide natural products has been extensively investigated because of the versatile pharmacological properties of these compounds (1-7). The type II polyketide synthase (PKS) is composed of dissociated enzymes that are used iteratively and are responsible for the elongation, cyclization, and modification of the polyketide chain ( Fig. 1) (3,4,8,9). The regiospecific cyclization of an acyl carrier protein (ACP)-linked linear poly-β-ketone intermediate is a key transformation catalyzed by type II PKSs. However, the enzymatic mechanism of cyclization remains poorly understood (10-14). Without such knowledge, the polyketide cyclization pattern cannot be predicted; a full understanding of this process at the molecular level is essential for future biosynthetic engineering efforts.In 2008, we reported the crystal structure of the first aromatase/cyclase (ARO/CYC) (TcmN ARO/CYC), which is a singledomain protein (15). On the basis of the structural analysis and mutagenesis results, we proposed that monodomain ARO/CYCs contain an active site and are capable of catalyzing polyketide cyclization and aromatization. Since then, we have performed structural and biochemical studies of two other monodomain ARO/CYCs: WhiE and ZhuI (Fig. 1) (16, 17). These studies provided strong evidence supporting our hypothesis that ARO/ CYC is the site of polyketide cyclization. However, many type II PKSs contain di-domain ARO/CYCs that have two seemingly identical domains (18-23). Why these enzymes require two domains (as opposed to just one) and how t...

The incorporation of nonacetate starter units during type II polyketide biosynthesis helps diversify natural products. Currently, there are few enzymatic strategies for the incorporation of nonacetate starter units in type II polyketide synthase (PKS) pathways. Here we report the crystal structure of AuaEII, the anthranilate:CoA ligase responsible for the generation of anthraniloyl-CoA, which is used as a starter unit by a type II PKS in aurachin biosynthesis. We present structural and protein sequence comparisons to other aryl:CoA ligases. We also compare the AuaEII crystal structure to a model of a CoA ligase homologue, AuaE, which is present in the same gene cluster. AuaE is predicted to have the same fold as AuaEII, but instead of CoA ligation, AuaE catalyzes acyl transfer of anthranilate from anthraniloyl-CoA to the acyl carrier protein (ACP). Together, this work provides insight into the molecular basis for starter unit selection of anthranilate in type II PKS biosynthesis.

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