Iron-sulphur protein catalysed [4+2] cycloadditions in natural product biosynthesis

Abstract

To the best of our knowledge, enzymes that catalyse intramolecular Diels-Alder ([4+2] cycloaddition) reactions are frequently reported in natural product biosynthesis; however, no native enzymes utilising Lewis acid catalysis have been reported. Verticilactam is a representative member of polycyclic macrolactams, presumably produced by spontaneous cycloaddition. We report that the intramolecular [4+2] cycloadditions can be significantly accelerated by ferredoxins (Fds), a class of small iron-sulphur (Fe-S) proteins. Through iron atom substitution by Lewis acidic gallium (Ga) iron and computational calculations, we confirm that the ubiquitous Fe-S cluster efficiently functions as Lewis acid to accelerate the tandem [4+2] cycloaddition and Michael addition reactions by lowering free energy barriers. Our work highlights Nature's ingenious strategy to generate complex molecule structures using the ubiquitous Fe-S protein. Furthermore, our study sheds light on the future design of Fd as a versatile Lewis acid catalyst for [4+2] cycloaddition reactions.

Fig. 1. Biosynthesis of verticilactams.

a Chemical structures of verticilactam (1) and verticilactams B (2) and C (3). b Biosynthetic gene cluster of 1 from S. spiroverticillatus JC-8444. c The proposed biosynthetic pathway for verticilactams. Solid arrows indicate a physiological pathway, whereas the dashed arrow shows an abiological reaction only observed by in vitro conversion. *VtlF possibly accelerates Michael addition reaction based on DFT calculations.

Fig. 2. Functional characterisation of VtlG (P450).

a AlphaFold2 predicted the structure of VtlG. The haem cofactor shown in cyan was modelled from PDB 6J85 as a template. b Compound 4 accumulated in the vtlG disruptant, and compound 5 nonenzymatically converted from 4 during isolation. Time-dependent conversion of 1 μM 4 into 1 in vitro in the presence of 0.5 μM VtlG, 0.1 mg/mL spinach Fd (c) or 0.1 mg/mL VtlF (d), 1 unit/mL spinach FNR, and 100 μM NADPH. The reactions were performed in 50 mM Tris-HCl (pH 7.5) at 30 °C, and the reaction products were monitored by UPLC–MS.

Fig. 3. Fds-catalysed [4 + 2] cycloadditions.

a AlphaFold2 predicted VtlF, MirFd, SceC, and TriM structures. The [3Fe–4S] cluster was modelled from PDB 1SJ1 and coordinated by three cysteine residues in each Fd. In vitro conversion of 1 μM 6 into 1 (b) and 1 μM 4 into 5 (c) in the presence of 50 μM spinach Fd, VtlF, MirFd, SceC, and TriM, respectively. The reactions were performed in 50 mM Tris-HCl (pH 7.5) at 30 °C for 30 min. The reaction products were monitored by UPLC–MS. Boiled spinach Fd was the negative control.

Fig. 4. Characterisation of Fe–S cluster as efficient Lewis acid.

a 1 μM 4 was incubated with 50 μM of apo-spinach Fd mutant, apo-MirFd mutant, reduced spinach Fd with [2Fe–2S](Cys)₄³⁻ cluster, and reduced MirFd with [3Fe–4S](Cys)₃³⁻ cluster, respectively. Fds were reduced with 1 mM sodium dithionite. b 1 μM 4 was incubated with 1 mM FeCl₃, 1 mM FeSO₄, 1 mM GaCl₃, 1 mM desferrioxamine (DFO), and 1 mM DFO with 1 mM FeCl₃. The in vitro reactions were conducted at 30 °C in 50 mM Tris-HCl (pH 7.5) for 30 min. c The crystal structure of the gallium-substituted Synechocystis sp. PCC 6803 Fd (SynFd), GaFd (PDB 5AUK). In vitro conversion of 1 μM 6 into 1 in 15 min (d) and 1 μM 4 into 5 in 30 min (e) in the presence of 200 μM SynFd and GaFd, respectively.

Fig. 5. Proposed cluster–ligand interaction mode and the energy diagram based on DFT calculations.

a In the [2Ga–2S + H](Cys)₄-L model, the reactant molecule L binds to the Ga_α atom through a fifth coordination bond and the Ga–S cluster is stabilised by protonation. The cysteine residues were simplified to methanethiolate groups (SMe). b DFT calculations for the conversion of 6 to 1. Activation-free energies (ΔG^‡) calculated at the M06-2X/SDD and 6-311 + G** (SCRF = CPCM, water) level of theory are given in kcal mol⁻¹ and distances in Å. The Diels–Alder [4 + 2] cycloaddition moves from left to right, followed by the Michael addition to the right. Energies and geometric structures of transition states (TS) and intermediates (int) with or without [2Ga–2S + H](Cys)₄-L model are given below and above the relative energy diagram.