Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases

Abstract

Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases.

Keywords: biosynthetic gene cluster; cyclases; enzyme screening; intra-molecular Diels-Alder reaction; polyketides.

Scheme 1

General structures of the two classes of spirotetronate polyketides.

Figure 1

Sequence similarity network (SSN) based on spirotetronate cyclase sequences described in the literature. Displayed is the SSN based on 286 unique amino acid sequences with an alignment score of 15, AbmU (yellow), AbyU (magenta), and other spirotetronate cyclases reported in the literature (pink) are highlighted. 17 putative spirotetronate cyclase sequences (black & blue) were selected and after homology modelling studies 12 (blue) sequences were selected for further investigation.

Figure 2

(A) Semi‐preparative HPLC trace of the scaled‐up Cyc15 assay with substrate 1 (t_R=12.2 min) after 3 h incubation. Displayed are the ELSD (dark grey) and the extracted mass of the positive total ion current of the substrate and product ([M+H]+345 m/z; light grey) 1. (B) Major products 2 and 3 isolated from in vitro assays with AbyU and Cyc15 respectively.

Figure 3

Analysis of product formation in biotransformations with cyclase library enzymes (1 mg/mL) or no enzyme and substrate (1 mM) by evaluating the total product formation compared to the formation of displayed diastereoisomer as percentage of the product fraction. Endpoints after incubations for 20 min with 1 were analysed by UPLC‐MS (QToF). The peak areas are resolved from the extracted mass of the total ion current of the substrate and the products (1‐3, [M+H]⁺ 345.170 m/z). Highlighting spirotetronate cyclases AbmU (yellow), AbyU (magenta), and others from literature (pink); putative spirotetronate cyclase (dark blue); non‐ spirotetronate cyclases (light blue); and the buffer control reaction (green, Ctr).

Figure 4

(A) Crystal structure of Cyc15 Chain A (8OF7) displayed as cartoon in blue with cavities in grey. (B) Residues of the active site are highlighted and labelled as sticks and (C) assigned from the docking studies to interact with product 3 binding. (D&E) Docked product 3 (green) from two perspectives is displayed in concert with the active site residues, highlighting the distance (yellow) of the cyclohexene ring towards Trp124.