Crystal structure and functional analysis of tetracenomycin ARO/CYC: Implications for cyclization specificity of aromatic polyketides

Abstract

Polyketides are a class of natural products with highly diverse chemical structures and pharmaceutical activities. Polyketide cyclization, promoted by the aromatase/cyclase (ARO/CYC), helps diversify aromatic polyketides. How the ARO/CYC promotes highly specific cyclization is not well understood because of the lack of a first-ring ARO/CYC structure. The 1.9 Å crystal structure of Tcm ARO/CYC reveals that the enzyme belongs to the Bet v1-like superfamily (or STAR domain family) with a helix–grip fold, and contains a highly conserved interior pocket. Docking, mutagenesis, and an in vivo assay show that the size, shape, and composition of the pocket are important to orient and specifically fold the polyketide chain for C9-C14 first-ring and C7-C16 second-ring cyclizations. Two pocket residues, R69 and Y35, were found to be essential for promoting first- and second-ring cyclization specificity. Different pocket residue mutations affected the polyketide product distribution. A mechanism is proposed based on the structure-mutation-docking results. These results strongly suggest that the regiospecific cyclizations of the first two rings and subsequent aromatizations take place in the interior pocket. The chemical insights gleaned from this work pave the foundation toward defining the molecular rules for the ARO/CYC cyclization specificity, whose rational control will be important for future endeavors in the engineered biosynthesis of novel anticancer and antibiotic aromatic polyketides.

Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides

“…The presence of this protein was intriguing because DABB proteins act as polyketide cyclases (e.g., TcmI cyclase) in Streptomyces species (21), although the bacterial cyclases show low sequence similarity to plant DABB proteins. The third candidate was a Betv1-like protein in the same protein family as the Streptomyces TcmN ARO/CYC polyketide cyclase (22). Several Betv1-protein family members function as enzymes in plant natural product biosynthesis (23).…”

The polyketide cyclase RemF from Streptomyces resistomycificus contains an unusual octahedral zinc binding site

“…Based on sequence comparisons the three putative cyclases essential for the biosynthesis of resistomycin, namely RemI, RemF and RemL have been assigned to three of the ten sequence families [6]. RemI is related to the cyclases represented by tetracenomycin aromatase/cyclase with the characteristic ''helix-grip" fold [10]. RemL and RemF belong to two different cyclase families that are not yet structurally characterized.…”

Evolution by Pervasive Gene Fusion in Antibiotic Resistance and Antibiotic Synthesizing Genes

“…Composite protein 1 (C1, Figure 1a,b) is 319 amino acids in length and contains an aromatase/cyclase-like domain and a phosphopantetheine binding domain (see Figure 1). The aromatase/cyclase domain participates in the diversification of aromatic polyketides by promoting polyketide cyclisation [36]. The phosphopantetheine binding domain serves as the attachment site of a 4'-phosphopantetheine prosthetic group.…”